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2009, Volume 40 Issue 4
06 August 2009 Previous Issue    Next Issue
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HSP40 and HSP70 inhibit the accumulation and toxicity of mutant huntingtin in N2a cells 2009, 40 (4):  517-521.  doi: 10.3969/j.issn.0529-1356.2009.04.001 Abstract ( )   Objective To study the effect of heat shock protein 40(HSP40) and heat shock protein 70(HSP70) on the aggregate formation of mutant huntingtin (htt) and its toxicity in N2a cells. Methods The effect of the over-expression of HSP40 and HSP70 alone or co-over-expression of them on aggregate formation of transfected mutant htt (containing 150 glutamine repeats, 150Q) in N2a cells was detected by fluorescence microcopy and Western blotting. Cell viabilities were detected by MTT assay. Reactive oxygen species (ROS) production was detected by colorimetric method. Results The over-expression of HSP40 and HSP70 alone, especially the co-over-expression of them, dramatically decreases the formation of 150Q htt aggregates in N2a cells. The numbers of aggregates in each group are as follows (EM>n/EM>=1 000): about 50% in the group of 150Q htt-expressing alone, about 12% in the group of overexpression of HSP40, about 15% in the group of overexpression of HSP70, about 5% in the group of co-over-expression of HSP40 and HSP70. The result of MTT assay shows that the over-expression of HSP40 and HSP70 alone, especially the co-over-expression of them, dramatically increases cell viabilities (EM>P/EM><0.01, EM>n/EM>=3) and reduces the production of ROS (EM>P/EM><0.01, EM>n=/EM>3). Conclusion HSP40 and HSP70 can increase cell viabilities of 150Q N2a cells via inhibiting the aggregates formation of mutant huntingtin and reducing oxidat Related Articles | Metrics

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Morphological and histochemical study of hippocampal dentate gyrus in reeler mice 2009, 40 (4):  522-526.  doi: 10.3969/j.issn.0529-1356.2009.04.002 Abstract ( )   P>Objective In order to investigate the influence of reelin deficiency on the hippocampal development, the histochemical characteristics of hippocampal pyramidal cells and granule cells of reeler mice were analyzed, therefore, reelin’s function would be better understood with more morphological evidences. Methods With immunofluorescent double labeling, the pyramidal cells, granule cells and mossy cells in hippocampi between wild type and reeler mice were labeled. Results The development and lamination of hippocampal cortical plate were in disorder. Pyramidal cells and granule cells dispersed, and moreover, granule cells proliferated rapidly and migrated into hilus, so that the bound between granule layer and hilus disappeared. Conclusion As a stop signal and regulatory signal, reelin plays important roles in neuronal migration of developing cortical plate, especially in the regulation of granule cell proliferation/P> Related Articles | Metrics

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Response of astrocytes and neurons in rat medullary visceral zone to the spared nerve injury 2009, 40 (4):  527-532.  doi: 10.3969/j.issn.0529-1356.2009.04.003 Abstract ( )   Objective To investigate the response of astrocytes and neurons in the medullary visceral zone (MVZ) to neuropathic pain induced by the spared nerve injury (SNI, the tibial nerve and the common peroneal nerve were sectioned, while the sural nerve was intacted). Methods The SNI operation and sham operation (only incised the skin of thigh, but the tibial nerve and the common peroneal nerve did not cut) were performed in adult male Sprague-Dawley rats. The paw withdrawal mechanical threshold (PWMT) was measured at 10,20,30 days after operation. By using single or double immunofluorescent staining method，we investigated and measured that the mean fluorescent intensity (MFI) of anti-glial fibrillary acidic protein (GFAP, a marker of astrocytes) signal in the astrocytes, the mean member of Fos/ GFAP double labeled astrocytes and Fos / tyrosine hydorxylase (TH) double labeled neurons in MVZ after operation. Results As compared with control or sham groups, SNI induced that the PWMT became significant sensitivity and peaked at 20 days after SNI. The activated astrocytes revealed an activated morphology, the MFI of anti-GFAP signal markedly strengthened, the mean number of Fos/GFAP double labeled astrocytes and Fos/TH double labeled neurons in the nucleus of the tract solitarius (NTS) and the ventral lateral medulla (VLM) increased significantly and peaked at 20 days after SNI. Conclusion The neurons and astrocytes in MVZ were sensitively activated by SNI. Related Articles | Metrics

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Effect of ginsenoside Rb1 on N9 cell activation induced by oxygen deficit 2009, 40 (4):  533-538.  doi: 10.3969/j.issn.0529-1356.2009.04.004 Abstract ( )   Objective To activate microglia N9 cell through the oxygen deficit, and to discuss the influence to the N9 cell by ginsenoside Rb1, laying the foundation for the basic study and the clinical medicine development. Methods Through ginsenoside Rb1 intervention, the cell morphology the proliferation ability were observed, ELISA, fluorescent probe DAF-FM DA, Griess the reagent examination, were used to measure TNF-α, the OSUB>2/SUB>SUP>-/SUP> output, the NO content change, chemiluminescence, the immunofluorescence method, and plastochondria membrane potential, were carried out to detect the cytochrome C content. Results Regardless of being preventive or medical gives, ginsenoside Rb1 can decline the NO,OSUB>2/SUB>SUP>-/SUP>,TNF-α high expression; and reduce the plastochondria membrane potential changing, the cytochrome C redistribution. Conclusion Ginsenoside Rb1 can decline N9 cell activation to a certain extent, reduce expression of the nerve toxic factor, and to stabilize mitochondrial membrane potential and distribution of cytochrome C. Related Articles | Metrics

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Morphological changes apoptosis and BDNF mRNA expression in the conus medullaris neurons in cauda equina compressed rats 2009, 40 (4):  539-545.  doi: 10.3969/j.issn.0529-1356.2009.04.005 Abstract ( )   Objective To investigate the morphological changes, apoptosis and brainderived neurotrophic factor mRNA（BDNF mRNA）expression in rat conus medullaris induced by cauda equine compression, and to discuss the possible mechanisms. Methods Ninety male SD rats were divided into 3 groups: normal control, sham operation，and cauda equine compressed. The animals were sacrificed and took samples in 30 min, 2 hours,4 hours,8 hours,1 day,3 days,1 week,2 weeks,3 weeks after cauda equine compression model were created. The morphological study concerned with cauda equina and conus medullaris was done under light microscope and transmission electron microscope. TUNEL staining and EM>in situ/EM> hybridization staining were used to investigate apoptosis and BDNF mRNA expression changes. The positive cells in 1mmSUP>2/SUP> were calculated,and the data were disposed through one way analysis of variance. Results Histological observation showed notable alteration of cauda equina and neurons in conus medullaris. The positive cells of TUNEL and BDNF mRNA EM>in situ/EM> hybridization staining increased in 8 hours and 4 hours after cauda equina compressed，and both reached the climax in 3days after cauda equina compressed. At the time 3weeks after cauda equine compressed, the positive cells were still much higher than that of the control groups. Conclusion The compression of cauda equina will result in neurons morphological changes and apoptosis in medullary cone,and cause central neuron unreversible injury. It might be one of the reasons why the prognosis is poor in cauda equina syndrome. Neurons and glial cells may prod Related Articles | Metrics

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Effect of APP 5-mer peptide on production and differentiation of cultured embryo hippocampal neural stem cells originated from SD rats 2009, 40 (4):  546-550.  doi: 10.3969/j.issn.0529-1356.2009.04.006 Abstract ( )   Objective To look for a small molecular neurotrophin which could promote production and/or differentiation of neural stem cells (NSCs). Methods 1.Embryo hippocampal NSCs were cultured in vitro. 2. The neurospheres were identified by antibodies of bromodeoxyuridine (BrdU), glial fibrillary acid protein (GFAP), microtubule associated protein 2 (MAP2) and Galactocerebroside (GalC). 3. The cells were divided into four groups, which were control group, FBS treated group, transsequence of APP 5-mer peptide treated group, APP 5-mer peptide treated group. The morphology of NSCs was observed in above four groups. 4. Cell counts, detection of clone information rate and diameter of clone were done to study the effect of APP 5-mer peptide on production of NSCs. 5. In addition, we also detected the MTT metablism rates in all groups. Results 1 The NSCs formed neurospheres and grew in floating. They were BrdU-positive. GFAP-positive, MAP2-positive and GalC-positive cells appeared after FBS were added into the medium. 2. The morphology of NSCs was not changed in APP 5-mer peptide treated group and trans-sequence group compared with the control group. 3. The cell number increased in APP 5-mer peptide treated group as compared with the control group. There were no apparent differences between the control group and the transsequence treated group. 4. The clone formation rate and diameters of neurospheres increased in APP 5-mer peptide treated group. 5. The MTT metabolism rate increased in APP 5-mer peptide treated group. Conclusion APP 5-mer peptide could promote the production of embryo hippocamal NSCs EM>in vitro/EM>. APP 5-mer peptid Related Articles | Metrics

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Expression of Nogo-A on the new born retinal ganglion cells and their axons of mouse embryos 2009, 40 (4):  551-554.  doi: 10.3969/j.issn.0529-1356.2009.04.007 Abstract ( )   Objective To investigate the expression of Nogo-A in the retinal ganglion cells (RGCs) and their axons of mouse embryos and its time course changes. Methods Sections of retinofugal pathway of C57 mouse embryos at different developmental stages were immunostained with Nogo-A specific antibody and observed by a confocal microscopy. The identity of Nogo-A positive cells was partially revealed by doublestaining together with Tuj-1 Results At the early stage of E12, Nogo-A was densely expressed in some radiallyorientated cells in retina. The immunopositive signals appeared in the cytoplasm, on the cell membrane and axons. The double-labeling together with Tuj-1, a neuronal marker, showed that nearly all the RGCs and their axons expressed Nogo-A protein. At the later stage of E13, the number of Nogo-A positive neurons in retina decreased dramatically. And those Nogo-A positive RGCs were specifically located in the ventricular part and the ciliary margin zone of the retina. At this stage, only a very few axons maintained their Nogo-A expression in the fiber layer of the retina, while most lost their Nogo-A distribution. When most RGCs had fully differentiated at E15, there was no detectable Nogo-A immunopositive staining in the retina and only a few retinal fibers were Nogo-A immunopositive. The similar expression patterns of Nogo-A was found in a few axons along the optic disc, optic stalk, optic chiasm and optic tract. Worthy of note, the retinal axons with Nogo-A distribution in the optic tract were exclusively found in the superficial area, where the newly arrived axons were traveling through during development. Conclusion The expression pattern and its time course change suggested that Nogo-A was an important protein expressed by the newly differentiated RGC neurons and their projecting axons, whilst the mature RGCs down-regulated their expression. Nogo-A in the new born RGCs might play some cellintrinsic roles such as de Related Articles | Metrics

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Effect of nicotine on the degeneration of dopaminergic neurons in the substantia nigra of PD rats 2009, 40 (4):  555-559.  doi: 10.3969/j.issn.0529-1356.2009.04.008 Abstract ( )   Objective To explore the effect of nicotine on the degeneration of dopaminergic neurons in PD rats. Methods Forty-five SD rats were randomly divided into three groups: PBS group (CON), normal saline + LPS ( NS ) group and nicotine +LPS ( NIC ) group. On 24 hours after LPS or PBS injection, inducible nitric oxide synthase ( iNOS ) protein expression was examined by immunoblotting. ON 14d after LPS or PBS injection, the numbers of tyrosine hydroxylase ( TH ) positive neurons and morphological changes of OX-42 positive cells in the substantia nigra(SN) were observed by immunohistochemistry. TH mRNA and TH protein expressions were examined by RT-PCR or immunoblotting. Results Compared with CON group, iNOS protein increased markedly 24 hours after LPS injection in NS group. TH positive neurons, TH mRNA and TH protein in the SN decreased remarkably 14 days after nigrainjiection. Most of microlglial showed big cell body with thicker and shorter processes. However, nicotine reversed the above changes, Compared with NS group, TH positive neurons, TH mRNA and TH protein in the SN increased remarkably in NIC. Besides, most microglia showed small cell body with slim and long processes. Conclusion Nicotine could prevent LPSinduced degeneration of DA neurons, probably because of that the pretreatment with nicotine blocks the activation of microglia and the expression iNOS protein. Related Articles | Metrics

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Effect of nicotine on the activation and resultant death of microglia induced by lipopolysaccharid 2009, 40 (4):  560-566.  doi: 10.3969/j.issn.0529-1356.2009.04.009 Abstract ( )   Objective To observe the effect of nicotine(NIC) on the activation and resultant death of microglia induced by LPS. Methods The animal model that exposed to chronic nicotine treatment was established and LPS was injected intraperitoneally to induce the activation of microglia. Furthermore, the CD11b-positive microglia in cerebral cortex, hippocampal and substantia ngra were observed through immunohistochemical staining. BV2 cells(Microglial cell line of mouse) were subcultured, simultaneously the following kits were used including CCK-8 kit assay for cell activity, Nitric oxide assay kit assay for NO release, RT-PCR assay for the iNOS,TNF-α,IL-1β,IL-6,COX-2,IRF-1,Caspase-11 mRNA expression, Western blotting assay for the protein expression of P-I-κB and Caspase-3. Results Nicotine suppressed the CD11b-positive microglia expression in cerebral cortex,hippocampal and substantia ngra induced by LPS; Nicotine inhibited the activation-induced cell death (AICD), attenuated NO release, reduced iNOS,TNF-α,IL-1β,IL-6,COX-2,IRF-1,Caspase-11 mRNA expression, decreased the protein expression including P-I-κB and Caspase-3 of BV2 cells. Conclusion Nicotine pretreatment can suppress the activation and resultant death of microglial cells induced by LPS, which suggests that nicotine Related Articles | Metrics

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Morphological, strutural and functional characteristics of cardiomyocytes from neonatal rats 2009, 40 (4):  567-572.  doi: 10.3969/j.issn.0529-1356.2009.04.010 Abstract ( )   Objective To investigate morphologic, structural and functional characteristics of cardiomyocytes from neonate rats and to set up a desirable technique for isolating and purifying the cardiomyocytes from neonate rats. Methods Using trypsin-digestion, mechanical separation, twice seedings and bromodeoxyuridine (BrdU)treatment, the cardiomyocytes were isolated and purified from the hearts of neonate rats at 1-7 days after birth. Shapes and spontaneous pulsation of the cells were viewed. The cells were identified with cardiac isoform of tropnin T(cTnT) immunostaining. Ultrastructural features of the cells were examined with EM>in situ/EM> transmission electron microscopy. Responses of the cells to adenine and isoprenaline were also examined. Results More than 95% cells isolated from the hearts of neonate rats are cardiomyocytes. The vital cells are more than 95%. Neonate rat cardiomyocytes include short columnar or rhabdoid cells and irregular cells. The most rhabdoid cells from the rats at 1-3 days after birth present the ultrastructural features of immature cardiomyocytes. The rhabdoid cells from the rats at 6-7 days after birth have some ultrastructural features of mature cardiomyocytes. Comparing with the cells at 1-3 days after birth, cTnT expression in the cells is slightly enhanced, the transverse striation was obvious. The irregular cells contain less bundles of myofilaments, the filaments are arranged irregularly. There are a few small cells which are in undifferentiated state. More than 80% cells show spontaneous pulsation at 72 hours after incubation. After treatment with adrenine and isoprenaline, the number of the cells with spontaneous pulsation increases and the intension of spontaneous pulsation is enhanced. The responses of the rhabdoid cells from the rats at 6-7 days after birth to adenine and isoprenaline are much stronger. Conclusion There are two kinds of neonate rat cardiomyocytes. They are different in ultrastructures, spontaneous pulsation and responses to adenine and isoprenaline. The cardiomyocytes from rats at 6-7 days after birth are suitable for experiments EM>in vitro/EM> as mature cardiomyocyte. The method set up in this experiment is desirable for culture of neonate rat cardiomyocytes. Related Articles | Metrics

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Comparative study on biological characteristics of marrow-derived cardiac stem cells from rats at different Ages 2009, 40 (4):  573-577.  doi: 10.3969/j.issn.0529-1356.2009.04.011 Abstract ( )   Objective To investigate the changes in morphology and senescence-associated markers of the marrow-derived cardiac stem cells (MCSCs) from rats at different ages and to explore the impacts of age on proliferation, survival and differentiation of MCSCs. Methods With single-cell cloning culture, MCSCs were selected from the bone marrow of young, adult and aged male SD rats respectively. Ultrastructural changes of the cells were viewed under a transmission electron microscope. The senescence-associated changes were examined with SA-β-galactosidase staining and reactive oxygen species(ROS) staining. Distribution of cell cycle of MCSCs from different age groups was evaluated with flow cytometric analysis. Rates of the survived and apoptotic cells were determined by Annexin V/PI double-labeled flow cytometric analysis and Hochest33342 staining. Differentiation of the MCSCs toward cardiomyocytes was induced with BMP-2. Expression of cardiac transcription factors and cardiac specific genes of the cells after induction were examined with RT-PCR. CTnT expression of the cells also be examined with immunocytochemistry. Results The nucleus/plasma ratio of the cells from aged rats decrease and there are some myelin bodies in the cells of aged group. With increasing of age, the MCSCs in S+GSUB>2/SUB>/M phase reduce, while β-galactosidase-positive cells and ROS-positive cells increase. Survival rate of the cells from aged rats is lower than that of the cells from young rats. At four week after induction with BMP-2, expression of Nkx2-5, GATA-4, cTnT mRNA and Cx-43 mRNA of the cells of young group increase significantly. In adult and aged group, expression of the cardiac transcription factors and cardiac specific genes is lower than that of the cells in young group. In immunocytochemical staining, cTnT expression of the cells in young group is stronger after induction with BMP-2. As compared with that of the cells in young group, cTnT expression of the cells in aged group is weak after induction. Conclusion With increasing of age, MCSCs show senescent changes, including their abilities of proliferation, survival and differentiation toward cardiomyocytes decrease. Related Articles | Metrics

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Human bone marrow mesenchymal stem cells could beinduced to differentiation by low dose sodium arsnite 2009, 40 (4):  580-584.  doi: 10.3969/j.issn.0529-1356.2009.04.012 Abstract ( )   P>Objective To study the effects of low dose sodium arsenite to human bone marrow mesenchymal stem cells(hBMSCs) differentiation during establishing of arsenic-resistant cell model. Methods hBMSCs were prepared in conventional method and continuously exposed to 1μmol/L sodium arsenite for ≥12weeks EM>in vitro/EM>. Forty-eight hours acute arsenite toxicity test was drived to assay if the cells acquired arsenic-resistance. The proliferation capacity of CAsE-hBMSCs was observed by the rate of colony formation.The expression of Oct-4 in CAsE-hBMSCs was assayed by RT-PCR and immunocytochemistry. The expression of ABCG2 in CAsE-hBMSCs was analyzed by RT-PCR. Results hBMSCs continuously exposed to 1μmol/L sodium asenite for ≥12 weeks exhibited dramatic resistance to acute arsenite toxicity. The LCSUB>50/SUB> for acute arsenite exposure in CAsE-hBMSCs was 35.59μmol/L versus 18.04μmol/L in control cells. Compared to control cells, the CAsE-hBMSCs didn’t show malignant proliferation ability. Expression of Oct-4 gene was positive in 4th, 18th passage hBMSCs and the hBMSCs induced by arsenite for 4 weeks but negative in CAsE-hBMSCs. The expression of Oct-4 protein was positive and weakly positive in 4th passage hBMSCs and CAsE-hBMSCs respectively, and the positive granules of Oct-4 distributed in cytoplasm. The expression of ABCG2 gene in CAsE-hBMSCs was obviously lower than that in control cells (EM>P/EM><0.001). Conclusion Human b Related Articles | Metrics

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Estrogen exerts the effect on stressinduced senescence of vascular smooth muscl cells EM>in vitro/EM> 2009, 40 (4):  585-589.  doi: 10.3969/j.issn.0529-1356.2009.04.013 Abstract ( )   Objective To explore the effects of estrogen on stressinduced senescence of vascular smooth muscle cells (VSMCs) and the underlying mechanisms. Methods The VSMCs of passage 23 cultured from female SD rats were induced into senescence by exposing to 150μmol/L HSUB>2/SUB>OSUB>2/SUB> in the presence or absence of different concentrations(10SUP>-10/SUP>mol/L10SUP>-8/SUP>mol/L) of 17β-estradiol (ESUB>2/SUB>). The expressions or activities of senescence associated marker DcR2, senescence-ssociated beta-galactosidase (SA-β-Gal), oncogene Ras and p21SUP>WAF1/SUP> were detected by flow cytometry, cytochemical staining, pulldown assay or Western blotting analysis. Results Flow cytometry analysis showed that in the physiological concentrations, ESUB>2/SUB> significantly inhibited the HSUB>2/SUB> OSUB>2/SUB>promoted highlevel expression of DcR2 of VSMCs in a dosedependent manner, with a highest inhibitive rate at 14.48%±0.6%（ESUB>2/SUB>=10SUP>8/SUP>mol/L;EM>P/EM><0.05, EM>n/EM>=3）; this inhibitive effect could be blocked by a ESUB>2/SUB> receptors inhibitor ICI 182,780. Cytochemistry staining showed that the rate of SA-β-Gal positive VSMCs induced by HSUB>2/SUB>OSUB>2/SUB> decreased in presence of 10SUP>-8/SUP>mol/L ESUB>2/SUB> (20.5%±1.4% vs 9.6%±0.9%;EM>P/EM><0.05, EM>n/EM>=9). Pulldown assay and Western blotting analysis revealed that administration of 10SUP>-8/SUP>mol/L ESUB>2/SUB> obviously reduced the HSUB>2/SUB>OSUB>2/SUB>induced Related Articles | Metrics

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Detection on mRNA exepression and activity of phosphodiesterase in rat pulmonary microvascular endothelial cells 2009, 40 (4):  590-593.  doi: 10.3969/j.issn.0529-1356.2009.04.014 Abstract ( )   Objective To study the main subtypes messenger ribonucleic acid（mRNA） and the basal enzyme activity of phosphodiesterase (PDE) in rat pulmonary microvascular endothelial cells (PMVECs） through the examination mRNA expression and activity of PDE EM>in vitro/EM>. The data were offered to reveal the relationship between PDE distributions, activity change and to accumulate data for the possibility of drug regulation of its functional alteration. Methods The cells were cultured with tissuesticking method; the gene expression of PDEs was detected by reverse transcript polymerase chain reaction (RT-PCR), and the activity of PDEs was calculated by cyclic nucleotides content change examined with high performance liquid chromatogram (HPLC) before and after the PDE reaction（EM>n/EM>＝3）. Results The PMVECs identified by cell immunofluorescence with polyclonal antibody of CD31 were dissociated and cultured, mRNAs of PDE1A, 1C, 2A，3A, 3B, 4A, 4D, 5A, 7A, 7B, 8A, 8B, 9A, 10A,11A were expressed in PMVECs, but there was no mRNA of PDE1B expressed in PMVECs. cAMP/cGMPPDE in the extent of 5-20μl had a Related Articles | Metrics

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Effect of Grp75 on the alteration of apoptosis related gene Bax and NF-κB induced by glucose deprivation 2009, 40 (4):  594-598.  doi: 10.3969/j.issn.0529-1356.2009.04.015 Abstract ( )   Objective To study the effect of glucose regulated protein 75(Grp75) on the alteration of Bax and NF-κB induced by glucose deprivation through the stably transfected PC12 cells with Grp75. Methods The cells of Grp75-overexpressing group and control group incubated in glucosefree DMEM medium for indicated time (6, 12, 24 and 48hours). The expression level of Grp75, Bax and the activity of NF-κB were determined by Western blotting, and the expression level of Bax was determined by semi-quantitative RT-PCR and Western blotting. Immunocytochemistry was performed using a conformation specific anti-Bax (6A7) antibody to detect the activation of Bax. Results The activation of Bax and the decline of NF-κB activity played important roles in the apoptosis of PC12 cells induced by glucose deprivation. Grp75 inhibited the apoptosis induced by glucose deprivation through inhibition of the activation of Bax and the decline of NF-κB activity. There was no change in Bax expression level under glucose deprivation in two groups. Conclusion The activation of Bax and the decline of NF-κB activity were associated with apoptosis of PC12 cells induced by glucose deprivation, and Grp75 provided protection to PC12 cells thro Related Articles | Metrics

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Hydrodynamics-based transgene directively into rat regenerating liver EM>in vivo/EM> 2009, 40 (4):  599-603.  doi: 10.3969/j.issn.0529-1356.2009.04.016 Abstract ( )   Objective To study the conditions and methods of hydrodynamicsbased transgene into rat regenerating liver EM>in vivo/EM>. Methods The solution with concentration 30mg/L genecontaining plasmid was injected into rat tail veins at a speed of 2ml/s, then partial hepatectomy (PH) was performed at different times before/after injection, finally the rat (g) and regenerating liver (g) were weighed, and the liver coefficient (LSUP>c/SUP>) was calculated. Out of 15 groups which are LSUP>c/SUP>±LSUP>c/SUP>*0%, *5%, *10%, *15%, *20%, *25%, *30%, *35%, the most suitable group was chosen as correction coefficient to calculate the most appropriate volume of plasmid solution which was injected into the regenerating liver at different recovery times, and at the same time, right lobe of liver was gathered to make frozen section, then observe and quantify the positive green fluorescent protein (GFP) rate at 488 nm excitation wavelength. Results Injection of either physiological saline or empty plasmid has no significant difference compared with control (only PH performance). The appropriate time of hydrodynamicsbased transgene is more than 12 hours before PH or anytime after PH. The solution volume of hydrodynamicsbased transgene into liver regenerating rat after PH is rat weight (g) ×9%×1/3×corresponding correction coefficient (Trc). Both vector and target gene have effect on the time and abundance of Related Articles | Metrics

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Genetic instability of the sFRP1 gene in hepatocellular carcinoma in Chinese people 2009, 40 (4):  604-608.  doi: 10.3969/j.issn.0529-1356.2009.04.017 Abstract ( )   Objective To examine loss of heterozygosity (LOH) and microsatellite instability (MSI) of locus D8S532 on chromosome 8 and their influence on the expression of sFRP1 in the hepatocellular carcinoma (HCCs), which may provide an experimental evidence for clarifying the mechanism of EM>sFRP1/EM> gene and tumor development. Methods DNA was extracted from formalin-fixed paraffin-embedded tissues. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and ordinary silver stain were used to study LOH and MSI of locus D8S532. Envision immunohistochemistry, Leica-Qwin computerized imaging system and Image-Pro PluS (IPP) version 4.5 professional imaging analysis software were used to assess the expression of sFRP1. Results The detection rates of LOH and MSI of locus D8S532 in the 36 specimens of HCC were 11.11% and 8.33% respectively. The down-regulation of sFRP1 was observed in 31 of 36 HCCs (86.11%) compared with non-carcinoma liver tissues, and the positive rate of sFRP1 protein of the HCCs was 52.78%( 19/36 ). The frequency of LOH was lower in the cases with positive expression of sFRP1 protein than those negative (0 vs 23.53%, P<0.05). Conclusion It was a common phenomenon that expression of sFRP1 protein is negative or low in Chinese with HCCs. The genetic instability of EM>sFRP1/EM> gene was one of causes, which lead to HCCs. LOH may play a major role in negative expression of sFRP1. Related Articles | Metrics

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Study on optimizing human acellular dermal matrix and fluorescence labeling the Co-cultured fibroblasts 2009, 40 (4):  609-613.  doi: 10.3969/j.issn.0529-1356.2009.04.018 Abstract ( )   Objective To optimize human acellular dermal matrix（ADM） and evaluate its biological characters. Methods Human skin was treated with hypertonic saline followed by NaOH maceration（group A）, hypertonic saline followed by sodium dodecyl sulfate (SDS) detergent（group B） or Dispase Ⅱ followed by Triton X-100（group C）, the resulting ADM were sectioned, and then were stained by special immunohistochemistry method. The cytotoxicity of them were evaluated by methyl thiazolyl tetrazolium (MTT) colorimetry and then cell compatibility was analyzed by cell culture；The optimized ADM resulted was choosen for use. Fibrablasts（FBs）were transfected with adenovirus vector encoding green fluorescent protein gene（Ad-GFP）and the growth of them on the optimized ADM was observed by fluorescent microscopy. Results Collagen and elastic fibers can still be observed in three kinds of ADM. The cells in dermis can be disintegrated both in group A and C, but not in group B. The cytotoxicity scores of the ADM prepared in group A and B were grade 0 or grade 1, while that of group C was more than grade 1The ADM prepared by NaCl-NaOH maceration had good biocompatibility. There was statistical difference in adhering number of NIH3T3 cells in group A and B. NIH3T3 cells grew well in group A and the re Related Articles | Metrics

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Relationship between EM>REG/EM> Iα gene expression and the clinicopathologic parameters in the patients with primary gastric carcinoma 2009, 40 (4):  614-620.  doi: 10.3969/j.issn.0529-1356.2009.04.019 Abstract ( )   P>Objective To investigate the relationship between expression of EM>REG/EM>Iα gene and its clinicopathologic parameters, the survival rate in the patients with primary gastric cancer. Methods Using RT-PCR after extracting RNA from paraffinembedded materials and immunohistochemical techniques, EM>REG/EM>Iα gene expression was investigated in 235 samples. And the relation among their results with the clinicopathologic parameters of primary gastric carcinoma was discussed in experiment by SPSS 13.0 statistic software. Results The positive EM>REG/EM>Iα mRNA was 78% (183/235) of primary gastric carcinoma and the positive rate of EM>REG/EM>Iα protein was 31.1% (73/235) in 235 patients with primary gastric carcinoma. EM>REG/EM>Iα gene expression in infiltrating tumors was found to be significantly higher compared with localized tumors (P<0.05). EM>REG/EM>Iα gene was closely linked to the infiltrative growth pattern, signet ring cell and poorly differentitated adencarcinoma. The incidence of venous invasion of EM>REG/EM>Iα genepositive differentiated adenocarcinoma was significantly higher than that of EM>REG/EM>Iα genenegative tumors. Moreover, the patients with EM>REG/EM>Iα genepositive differentiated adenocarcinoma were found to have a significantly poorer prognosis as compared with EM>REG/EM>Iα genenegative tumor patients. Conclusions The results of the experiment demonstrated that the expressio Related Articles | Metrics

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Effect of cold stress on the expression of Huntingtin-associated protein 1 in the rat adrenal medulla 2009, 40 (4):  621-624.  doi: 10.3969/j.issn.0529-1356.2009.04.020 Abstract ( )   Objective To observe the ultrastructure location of Huntingtin-associated protein 1（HAP1） in rat adrenal medulla and the effect of cold stress on the expression of HAP1 in rat adrenal medulla. Methods Fourteen healthy male Wistar rats were used in the present study and among them two rats were used for immune electron microscopy and twelve rats for cold stress experiment. In the cold stress experiment, animals were divided into control and cold groups randomly with six rats in each group. During the experiment, rats were housed in a room at the temperature of 4℃ for 12 hours and then immunohistochemistry and Western blotting were used to measure the expression of HAP1 in adrenal medulla. Results By using the immune electron microscopy, the results showed HAP1 was located on the external membrane of secretory vesicles of adrenal medulla and their membranous organelles. The expression of HAP1 in adrenal medulla of cold group significantly decreased as compared with that of control group (EM>P/EM><0.01). Conclusion HAP1 might be related with the adrenal medulla cells endocrine granula and involved in the transmission and release of adrenaline or noradrenaline in secreto Related Articles | Metrics

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Expression of RhoGDIα in aortae of hypertensive rats 2009, 40 (4):  625-629.  doi: 10.3969/j.issn.0529-1356.2009.04.021 Abstract ( )   Objective To evaluate the role of angiotensin Ⅱ(AngⅡ) signal passway on the expression of Rho GDP dissociation inhibitor alpha (RhoGDIα) in hypertensive rats. Methods Protein and mRNA expressions of RhoGDIα in aortae of 4, 12 and 18 weekold spontaneously hypertensive rats (SHR, EM>n/EM>= 4) and Wistar Kyoto rats (WKY, EM>n/EM>= 4) were examined by Western blotting and realtime PCR. Aortas from SHR and WKY were analyzed using immonuchemical staining to locate the RhoGDIα in the aorta. The RhoGDIα expression in aorta of hypertensive rat model of aorta coarctation (ACR, EM>n/EM>= 6) was also analyzed using Western blotting. Furthermore, The effect of mechanical strain at 10 % elongation on expression of RhoGDIα in vascular smooth muscle cells (VSMCs) in the presence or absence of L-158809, an antagonist for AngⅡ type 1 receptor, was also evaluated by Western blotting. Results No significant difference of RhoGDIα expression was found between SHR and WKY at 4week-old and 12-week-old. However, in 18-week-old group, RhoGDIα was significantly highly expressed in SHR than that of WKY at both mRNA and protein levels. RhoGDIα was located in the media of the aorta. Expression of RhoGDIα protein was upregulated in aortas of ACR at 2 and 4 weeks as compared with the cont Related Articles | Metrics

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Early development of the sinus venosus and the cardiac conduction system in human embryonic heart 2009, 40 (4):  630-636.  doi: 10.3969/j.issn.0529-1356.2009.04.022 Abstract ( )   Objective To investigate the early development of the sinus venosus and the cardiac conduction system (CCS) of human embryonic hearts. Methods Serial transverse sections of 29 human embryonic hearts from Carnegie stage 10 to Carnegie stage 16 (C10-C16) were stained immunohistochemically with antibodies against α-smooth muscle actin(α-SMA),α-sarcomeric actin(α-SCA) and desmin ( DES ). Results During C12 and C13, the sinus venosus formed by confluence of systematic veins at the caudal end of the pericardial cavity could be recognized in the mesenchyme of primitive transverse septum. The mesenchymal cells of the sinus venosus gradually differentiated into α-SCA positive cardiocyocytes. At C14, the sinus venosus was within the pericardial cavity due to expansion of the pericardial cavity and incorporated into the right atrium. Differentiation of DES positive conductive cardiomyocyte was initiated in the right wall of atrioventricular canal of C10 embryonic heart and with the development, extended towards the myocardium of the interventricular sulcus to form His bundle, left and right bundle branches as well as the ventricular trabecular myocardium. In the atium, the strong expression of DES was first detected in the dorsal wall of C11 atrium. At C13, unique myocardial band showing α-SCA, α-SMA and DES expression in the left dorsal wall of the sinus venosus were found to be continuous with the basal wall of left atium and the dorsal wall of the atrioventricular canal, this band might be related to the development of conduction system from sinoatrial node to atrioventricular canal. During C14 to C16, primary conduction pathway of atria with strong DES expression was formed that extended from sinoatrial node along venous valves, DES positive myocardium in the dorsal and ventral walls of the atria to the right atrioventricular canal, respectively. Conclusions The mesenchyme of the primitive transverse septum is the heart forming field of human embryos responsible for formation of sinus venosus myocardium, cardiomyocytes are differentiated from mesenchymal cells in the primitive transverse septum and progressively added to the venous pole of the heart tube to form myocardial sinus venosus. The differentiation of CCS of the early human embryo initiates in the atriov Related Articles | Metrics

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Influence of quercetin and bornyl acetate on ratio of IFN-γ/IL-4 and macrophages in uteri of LPS-induced abortion mice 2009, 40 (4):  637-641.  doi: 10.3969/j.issn.0529-1356.2009.04.023 Abstract ( )   Objective To investigate significance of interferon-γ/interleukin-4(IFN-γ/IL-4) and macrophages in uterus in early embryo loss (or resorption), and to elucidate the anti-abortive effect and the immunological modulation at maternal-fetal interface with quercetin and bornvl acetate. Methods Lipopolysaccharide (LPS)（0.10μg/mouse）was injected via tail vein in order to induce abortion in 7-day-gestation mice which received quercetin and bornvl acetate at days 4-7 of gestation. Levels of IFN-γ and IL-4 in uterus lysate supernatant were measured using enzyme-linked immuno-absorbent assay (ELISA), and uterine macrophages of each group (EM>n/EM>=10) were detected by immunohistochemistry. Results The amount of macrophages was much higher in the uteri of LPSinduced abortion mice than that in control mice. The abortion rate of mouse declined to certain level. The therapy of quercetin combined with bornyl acetate reversed LPS-induced abortion. Conclusion The increase of IFN-γ/IL-4 and the amount of macrophages in the LPStreated mouse uterus is associated with the embryo loss, and que Related Articles | Metrics

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Research on histological structure of autofluorescence of EM>Dugesia japonica/EM> 2009, 40 (4):  642-646.  doi: 10.3969/j.issn.0529-1356.2009.04.024 Abstract ( )   Objective Hermaphroditic planarians possess a very important position in the systematic evolutionary history of animal, as they are capacity of complete regeneration. Hence, the research on histological structure of autofluorescence has been carried out to provide a crucial insight into the developmental and regenerative biology. Methods Part of histological structure of planarian (EM>Dugesia japonica/EM>) was revealed with HE method, Masson method and Van Gieson method. Their autofluorescence was observed with ultraviolet. There were six planarians in each stained group and the autofluorescence group. Results Epidermis, outer epidermis of pharynx, protonephridium, intestine, the photoreceptor cells and longitudinal nerve cords, all radiated blue autofluorescence. The epithelial dissociation side of copulatory bursa radiated yellow autofluorescence, its middle part radiated blue autofluorescence, its fundus side radiated weakly blue autofluorescence. Testis could hardly give off autofluorescence. Pigment cells of eyepot could not give off autofluorescence. Conclusion The research on configuration and autofluorescence of planarian eye may offer help for the study of origin and evolutionary law on eye of invertebrate. Related Articles | Metrics

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Immunohistochemical study for the expression of LHR and VEGFon the ovary of mice during peri-implantation 2009, 40 (4):  647-650.  doi: 10.3969/j.issn.0529-1356.2009.04.025 Abstract ( )   Objective To explore the biological effects of the luteinizing hormone receptor （LHR） and vascular endothelial growth factor （VEGF） on the ovary of mice during periimplantation. Methods The immunohistochemistry SABC method and image analysis were used to study the distribution and changes of the LHR and VEGF in Kunming mouse(EM>n/EM>=28) ovary during estrous，pregnancy of day 1, day 4 and day 6 stage. Results The expression of LHRimmunoreactive substance and that of VEGF-immunoreactive substance had the same distribution and changes. Compared with other groups,the level of LHR-immunoreactive substance and that of VEGF-immunoreactive substance increased highly on the stroma cells around larger growing follicles in estrous group (P<0.05). Along with the pregnancy, the positive immunostaining for LHR and VEGF increased gradually on the granulosa lutein cells, and reached the highest level on day 6 of pregnancy. Positive immunostaining for LHR or VEGF on some endothelia and blood cells were observed in day 1 of pregnancy or estrous group respectively. Form day 1 of pregnancy, the theca cells had positive immunostaining for LHR. Conclusion The expression of LHR and VEGF is closely r Related Articles | Metrics

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Effect of regional gene therapy with bone morphogenetic protein 7 producing mesenchymal stem cells on spinal fusion in rats 2009, 40 (4):  651-655.  doi: 10.3969/j.issn.0529-1356.2009.04.026 Abstract ( )   Objective To evaluate effectiveness of EM>ex vivo/EM> adenoviral gene transfer creating human bone morphogenetic protein 7producing mesenchymal stem cells (Ad-hBMP-7 cells) compounded with nano-hydroxyapatite/collagen(NHAC) composite to induce spinal fusion in a rat posterolateral spine fusion model. Methods Mesenchymal stem cells were cultured and transduced by Ad-hBMP-7. Then Ad-hBMP-7 cells were cocultured with NHAC. Totally 56 Wistar rats were divided into four groups. Intertransverse spinal arthrodesis was attempted in four groups of Wistar rats with autogenous bone (group A); NHAC(group B); NHAC and BMSCs (group C); Ad-hBMP-7 cells and NHAC (group D). Each specimen underwent plain radiography, manual palpation and histological analysis. Results All spines in group D had fusion. In contrast, none of the spines in group A and group B had fusion. Three of ten rats in group C achieved fusion at the 12th week post-operatively.Conclusion BMP-7-producing mesenchymal stem cells compounded with NHAC composite may induce intertransverse fusion in the rat spine model. Related Articles | Metrics

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Morphological changes of rat pancreatic tissue induced by ligation of thoracic duct 2009, 40 (4):  656-659.  doi: 10.3969/j.issn.0529-1356.2009.04.027 Abstract ( )   Objective To observe the morphological changes of pancreatic tissue of thoracic duct ligated rats in fine and ultrastructural levels, and to determine whether lymph block animal model can affect pancreatic islet amyloid polypeptide（PIAP）deposit in rat pancreas. Methods At the 6th month after the operation, some pancreatic tissue sections of 16-month-old experimental rats were embedded in paraffin wax and stained with HE and Congo red; immunohistochemical staining was performed on some frozen sections, which were then observed with light microscope; transmission electron microscope (TEM) specimen preparation and observation were performed on other samples. Results HE and Congo red stained sections showed that the pancreatic glandular lobule space was widened, with significant connective tissue hyperplasia, and fat accumulation when the islet was stained indistinctly or vermeil and tissue space was broadened. The sections with immunohistochemical staining displayed the pancreatic islet as well as the tissues around it were stained into dark brown being positive with PIAP antigen. TEM observation showed the pancreatic glandular interlobule space was widened, while blood vessels and enlarged lymphatic vessels were visible; within widened pancreatic islet interstitial space, a great quantity of lipid droplets and some collagen fibril structures could be seen.Conclusion The ligation of thoracic duct can contribute to pancreatic lymph draining block, lymphagiectasis, connective tissue space and interstitial space widening, fat accumulation, and PIAP deposit in rat pancreas. These structural changes may affect the function of pancreatic islets. Related Articles | Metrics

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Contrasted study between thin coronal sectional anatomy of the pineal region and MRI image 2009, 40 (4):  660-665.  doi: 10.3969/j.issn.0529-1356.2009.04.028 Abstract ( )   Objective To investigate the morphology and relationships with the adjacent structures in the pineal region on the thin sections and to provide anatomic data for imaging diagnosis and surgical treatment of diseases in this region. Methods By CT and MRI examination, one normal head specimen was selected for this study. Using the computerized freezing milling technique, the specimen was sliced from anterior to posterior. The EM>in vivo/EM> MR images were obtained from ten normal Chinese male adult volunteers using a 3.0 T GE scanner. The base lines of the sectioning and the MR scan were perpendicular to the AC-PC line. Then primary sections were contrasted with the corresponding MR images. Results By the appearance of the pineal peduncle and the disappearance of the pineal gland, the pineal region could be divided into three parts from anterior to posterior, and the shape changed from an inverted triangle to a trapezoid and a triangle gradually. The first interspace was getting wider in the anterior and middle parts of the pineal region, while in the posterior part of the pineal region, it was getting narrower and disappeared finally. From anterior to posterior, the bilateral internal cerebral veins were always in the midline of the pineal region and descended gradually.Conclusion By the computerized freezing milling technique, the anatomic details and adjacent relationships of the pineal region could be exhibited clearly in the thin serial sections, which could help the imaging diagnosis and surgical treatments for minute diseases in this region. Related Articles | Metrics

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Microanatomy and virtual anatomy of retrosigmoid approach 2009, 40 (4):  666-670.  doi: 10.3969/j.issn.0529-1356.2009.04.029 Abstract ( )   Objective To explore the related structures of retrosigmoid approach by microanatomy and virtual anatomy and provide a reliable approach with more morphologic data. Methods Twenty sides of 10 adult cadaveric heads were dissected to simulate retrosigmoid approach under the microscope. The neurovascular structures of pontocerebellar trigone were observed, and the related structures were simultaneously measured based on the junction of superior petrosal sinus and sigmoid sinus and internal acoustic pore. The internal auditory canal was opened by drilling the margin of the internal auditory meatus and its structures were watched. With the help of Dexotroscope system, the data of five patients’ CT and MRI were applied to reconstruct and anatomize the structure of retrosigmoid approach. Results It was found that the distance from the junction of superior petrosal sinus and sigmoid sinus to the trigeminal nerve was (38.50±2.64)mm, to the acoustic-facial bundle (27.80±2.25)mm, to the glossopharyngeal nerve (32.70±2.11)mm, to the hypoglossal nerve (44.30±2.05)mm, and the distance from internal acoustic pore to the trigeminal nerve was (5.68±1.55)mm, to the abducent nerve (13.80±1.81)mm, to the tentorium of cerebellum (5.00±0.66)mm, to the glossopharyngeal nerve (6.34±1.24)mm. The pontocerebellar trigone was divided into the anterior compartment, the middle compartment, the posterior compartment built on the acoustic-facial bundle and the glossopharyngeal nerve. Their structures were displayed after drilling the margin of the internal auditory meatus. Dexotroscope system clearly displayed asterion, the angle of transverse and sigmoid sinus, jugular foramen, internal acoustic pore, basilar artery and its branches, and theirs spatial relationship.Conclusion. The three compartments of the pontocerebellar trigone are helpful to understand the feature of the neurovascular layer, the measurement is favorable to quantize the relation of the related structures and to judge the space of each compartment. Recognizing the anatomical marker of internal acoustic pore can support preservation of the inner structures. Virtual anatomy of Dexotroscope system can display local anatomical structure respectively. Both microanatomy and virtual anatomy have their advantages and disadvantag Related Articles | Metrics

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Expression of endocytic receptor megalin/cubilin in embryonic mouse kidneys 2009, 40 (4):  671-674.  doi: 10.3969/j.issn.0529-1356.2009.04.030 Abstract ( )   Objective To investigate the expression of endocytic receptor megalin and cubilin BR>in in the developing mouse kidneys, and the correlation between the expression and the development of the renal tubules. Methods Expression of megalin and cubilin in developing mouse kidneys was examined at different embryonic days (E) using immunohistochemistry. Meanwhile, the ultrastructure of developing proximal tubules related to endocytosis was observed at transimission electron microscope level. Results At E9.5, megalin and cubilin were coexpressed in apical plasma membrane of the mesonephric ducts and mesonephric tubules. From E11 to E18, in the metanephros, the expression of both receptors were seen at the free surface and apical plasma of uretic bud, but weakly in all renal tubules of S shaped body at early differentiatial stage. With the mature of proximal tubule development, they were both confined to the brush border and the apical plasma of the proximal tubules in juxtamedullary cortex.Conclusion The endocytic receptors, megalin and cubilin are expressed in apical part of nearly all renal tubule epithelia in early development, and confined to free surface of mature proximal tubules, suggesting that with the mature of proximal tubules, the two receptors are generally involved in collaborating to facilitate, the reabsorption of ultrafiltration. BR> Related Articles | Metrics

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A reliable method to induce neural stem cells directed differentiating into neurons EM>in vitro/EM> 2009, 40 (4):  675-679.  doi: 10.3969/j.issn.0529-1356.2009.03.031 Abstract ( )   Objective To introduce a reliable method to induce neural stem cells directed differentiating into neurons EM>in vitro/EM>. Methods The rat neural stem cells were cultured in selected serum-free medium. After cultured for 2, 3 passages，the neurospheres or single-cells isolated from neurospheres were cultured in conditioned medium to induce directed differentiating into neurons. Besides morphological observation of those cells under inverted microscope, NSE immunocytochemistry was carried out to detect the differentiating ratio of those cells from neural stem cells into neurons. Results It was shown that the conditioned medium could induce neural stem cells differentiating into neurons effectively in both neurospheres culture mode or single-cells culture mode. Furthermore, compared with the neurospheres culture mode, the single-cells culture mode showed more synchronous in the differentiation process and higher in the percentage of NSEpositive cells. Conclusion This method can induce neural stem cells directed differentiating into neu Related Articles | Metrics

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Compararative investigation of proliferation of lymphocytes stimulated by human umbilical cord blood mesenchymal stem cells with CFSE and MTT 2009, 40 (4):  680-684.  doi: 10.3969/j.issn.0529-1356.2009.04.032 Abstract ( )   Objective To compare the sensitivities of the stimulation effect of human cord blood mesenchymal stem cells(CB-MSCs) and CB-MSCs of neuronal differentiation to lymphocytes(LCs) detected with carboxyfluorescein didcetate(CFSE) and MTT. Methods To prepare LCs from SD rat and divided into four group stimulating cells: 1. CB-MSCs; 2. Dif-CB-HSCs; 3. SH-SY5Y（positive control）; 4. Auto-LC（negative control）.Stimulating cells were respectively Co-cultured with LCs. The proliferation of LCs was detected with MTT and CFSE (EM>n/EM>=3). Results CB-MSCs and Dif-CB-HSCs stimulated LCs to proliferate more weakly than positive control detected with MTT and CFSE. The quantity of proliferation of lymphocytes Co-cultured with CB-MSCs and Dif-CB-HSCs were higher than that of Auto-LC detected with CFSE. But MTT OD value of CB-MSCs and Dif-CB-HSCs was a little lower than that of Auto-LC. Statistical analysis results showed no significant difference.Conclusion CFSE can reflect proliferation status of lymphocytes better than MTT. CFSE shows mor Related Articles | Metrics

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Cryopreservation of mouse spermatogonial stem cells 2009, 40 (4):  685-689.  doi: 10.3969/j.issn.0529-1356.2009.04.033 Abstract ( )   Objective To explore the conditions and methods for cryopreservation and proliferation of mouse spermatogonial stem cells （SSCs）. Methods SSCs were isolated from six-day-old Kunming mouse using two-step enzymatic digestion and Percoll discontinuous density gradient centrifugation. Cells were frozen with different freezing medium and cooling rate. After thaw, they were cultured in mimimum essential medium alpha (MEMα) supplemented with 10% fetal calf serum (FCS) and 100μg/L glial cell line-derived neurotrophic factor (GDNF). The survived and proliferating SSCs were examined by WST-8 colorimetric assay. Alkaline phosphatase and reverse transcription-polymerase chain reaction (RT-PCR) were performed to confirm if the cultured 96 hours germ cells were still stem cells. Results The best method to cryopreserve SSCs is using cryoprotector containing 10% dimethyl sulfoxide(DMSO), 10% FCS, 0.07mol/L sucrose and 1℃/min cooling rate, and the viability of cells in this method is more than 84%; Although the cell viability in nonprogrammed freezing method is less than that in the programmed freezing method, it is a simple and effective cropreservation method for mouse SSCs. What is more, the anchoring time of SSCs in this method is 8-12 hours after thaw, SSCs begin to proliferate 24 hours later, and rapid proliferation appears on the 48 hours, colonies are composed by 20-25 cells in 96 hours, when SSCs proliferated nearly 5 times.Conclusion The culture condi Related Articles | Metrics