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2007, Volume 38 Issue 2
06 April 2007 Previous Issue    Next Issue
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CHANGES IN mRNA FOR NEURONAL NITRIC OXIDE SYNTHASE ASSOCIATED PROTEIN NIDD FOLLOWING PERIPHERAL NERVE INJURY 2007, 38 (2):  129-133.  doi: Abstract ( )   Objective To study the localizations and changes of mRNA for nNOSinteracting DHHC domaincontaining protein with dendritic mRNA (NIDD) in normal and injured peripheral nerves. Methods Realtime fluorescence quantitative PCR (FQPCR) and a combination of in situ hybridization and indirect immunofluorescence were used to detect the localizations and changes of mRNA for nNOS and NIDD. Results It was found that nNOS and NIDD were mainly localized in Schwann cells (SCs) of normal sciatic nerve, and the expression peaked in crushed sections of sciatic nerve in the 2nd week after injury and in proximate or distal transected stumps in the 1st week after injury respectively. Expressions of mRNA for nNOS and NIDD were detected in the perinuclear plasm of primary SCs as well. Conclusion The increase in mRNA for NIDD in SCs may Related Articles | Metrics

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CHANGES IN THE EXPRESSION OF OREXIN AND ITS RECEPTORS IN THE RAT HYPOTHALAMUS DURING PREGNANCY, PARTURITION AND LACTATION 2007, 38 (2):  134-138.  doi: Abstract ( )   P>Objective To investigate the possible role of orexin in reproduction and examine the changes in the expression of orexin and its receptors (OX1R, OX2R) in the rat hypothalamus during pregnancy, parturition and lactation. Methods The expressions of preproorexin (preproOX), orexinA, OX1R, and OX2R in the rat hypothalamus during pregnancy, parturition, and lactation were evaluated by competitive reverse transcriptionpolymerase chain reaction (RTPCR) and immunohistochemistry assay. Results OrexinA immunoreactive (ir) neurons and the OX1R subtype in neuronal cell bodies were mainly located in the lateral hypothalamic area (LHA) as well as in the magnocellular neurons of the paraventricular and supraoptic nuclei respectively in pregnant and lactating rats. The preproOX and OX1R mRNA levels on the 1st day of lactation were significantly higher than that during late pregnancy and lactation. No significant changes of OX2R expression were observed during the various reproductive phases. Conclusion Orexin might be involved in regulating reproductive func Related Articles | Metrics

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THE RELATIONSHIP BETWEEN GLUTAMATE INPUTS FROM THE SUBICULAR COMPLEX AND THALAMOCORTICAL PROJECTION NEURONS IN THE ANTERIOR THALAMIC NUCLEUS OF THE RAT 2007, 38 (2):  139-143.  doi: Abstract ( )   Objective To examine the synaptic structure and glutamatergic transmitter of the pathway linking the anterior thalamic nucleus(ATN) and the subicular complex. Methods The HRP tracing and post embedding immunogold technique were used. Results In the anterior thalamic nucleus, anterograde HRP labelled terminals contained clear round synaptic vesicles and several mitochondria, and formed asymmetric synaptic contacts with HRPlabelled or nonHRP labelled dendrites. The highest densities of immunogold particles following glutamate immunostaining were found in HRPlabelled terminals and similar axon terminals devoid of HRP reaction product, They formed asymmetric synaptic contacts(Gray type Ⅰ) with dendrites. The average density of those immunogold particles was more than 3 times higher than that of the gold particles in the dendrites of their contacts and over 6 times higher than that of the particles in the terminals that formed symmetric synapses (Gray type Ⅱ). In two serial GABA immunogold reactive sections, Gray tpye Ⅱ terminals were heavily labeled whereas Gray type Ⅰterminals displayed a very slight labelling. In glutamate immunogold reactive sections, Gray type Ⅱ terminals were slightly labeled. A GABA positive terminal which formed symmetric synapses with HRP-labeled dendrites and the terminal which formed asymmetric synapses converged on the same dendrite. Conclusion The terminals of projection neurons in the pathway linking the anterior thalamic nucleus and the subicular complex are glutamatergic. In anterior thalamic nucleus corticothalamic projection neuron term Related Articles | Metrics

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THE STUDY OF THE DIFFERENTIATION OF NSCs MODIFIED WITH ISLET-1 GENE INTO CHOLINERGIC NEURONS 2007, 38 (2):  144-147.  doi: Abstract ( )   Objective To explore whether Islet-1 gene of the rat could induce NSCs to differentiate into cholinergic neurons. Methods NSCs were transduced with Islet-1 recombinant retroviral expression vector. The protein expression of Islet-1 gene in NSCs was detected by immunofluorescence histochemical method. The ability of NSCs to differentiate into ChAT positive cells was observed in vivo and in vitro. Results The numbers of ChAT positive cells were significantly increased in the group of NSCs modified with Islet-1 compared with the control group in vitro. NSCs mdified with Islet-1 could differentiate into ChAT positive cells when they were grafted into the corpus striatum of the adult rat brain. Conclusion Islet-1 gene could induc Related Articles | Metrics

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EXPERIMENTAL STUDY ON DIRECTIONAL DIFFERENTIATION OF HUMAN UMBILICAL CORD BLOOD(HUCB) STEM CELLS INTO DOPAMINERGIC NEURONS 2007, 38 (2):  148-152.  doi: Abstract ( )   Objective To investigate and compare some conditions which could induce human umbilical cord blood(HUCB) stem cells to differentiate into dopaminergic neurons, and probe the optimal condition for the directional differentiation of HUCB stem cells. Methods HUCB stem cells were isolated and cultured in vitro, and the cell surface markers were detected by flow cytometry, and when the cells were cocultured with EGF+bFGF, ATRA, ATRA+EGF+bFGF, striatal conditioned medium(CM)、striatal astrocytes conditioned medium(CM) respectively, the morphological character was observed by inverse phasecontrast microscopy. The expression of tyrosine hydroxylase(TH) was detected by immunofluorescence staining method. Results The comparison of the results of HUCB stem cells which directly differentiated into dopaminergic neuron showed that the differentiation potential of striatal astrocytes CM group>striatal CM group>ATRA+EGF+bFGF group>ATRA group>EGF+bFGF group>control group.Conclusion Compared with other conditions, the striatum has a more distinct inductive effect on HUCB stem cells to differentiate into dopaminergic neurons, and the inductive effect m Related Articles | Metrics

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THE EXPRESSION OF NESTIN AND SSEA-I IN THE RAT TYPE-2 ASTROCYTES 2007, 38 (2):  153-157.  doi: Abstract ( )   Objective To investigate whether type-1 and type-2 astrocytes (T1A, T2A) express the characteristic molecules of neural stem cells. Methods Purified cultures of cortical O-2A progenitor cells, T1A and T2A were prepared from neonatal rats. The expression of nestin and stage specific embryonic antigens1 (SSEA-1) were detected by immunocytochemistry labeling. O-2A progenitor cells, TIA and T2A were cultured in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) for 10 days. Then the morphological changes of the above cells were examined. Results Expression of nestin was observed in O-2A progenitor cells and T2A, while T1A was negative for nestin immunostaining. Expression of SSEA-1 was only detected in T2A. After treated with bFGF and EGF, T2A changed into neurosphereslike morphology and could be passaged continuously. The spheres were also positive for nestin immunolabeling. After plated onto PLLcoated substrates for 10 days, cells with typical morphologies of neuron, astrocytes and oligodendrocytes were observed to migrate from the spheres. Conclusion Nestin and SSEA-1 were expressed differently in T1A and T2A. T2A induced by extracellular signals may acquire some properties of the multipotent neura Related Articles | Metrics

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A MORPHOLOGICAL STUDY OF CONNECTIONS BETWEEN ENKEPHALIN IMMUNOREACTIVE TERMINALS AND γ-AMINOBUTYRIC ACID IMMUNOREACTIVE NEURONS IN THE ROSTRAL PORTION OF THE NUCLEUS TRACTUS SOLITARIOUS OF THE RAT 2007, 38 (2):  158-162.  doi: Abstract ( )   Objective To observe the connections between enkephalin immunoreactive (ENK-ir) terminals and γ-aminobutyric acid immunoreactive (GABA-ir) neurons in the rostral portion of the nucleus tractus solitarious (rNTS) of the rat. Methods Doubleimmunofluorescent labeling method and the preembedding immunohistochemical staining combined with immunogold particles labeling technique for electron microscopic detection were used in the present study. Results Under the laser scanning confocal microscope, dense ENK-ir fibers and terminals and some GABA-ir neurons were observed in the rNTS. Close contacts between ENK-ir terminals and GABA-ir cell bodies were also detected. By using the electron microscopic technique, ENK-ir reaction products were found to be mainly localized on the surface of round clear vesicles and densecored vesicles in the axon terminals. ENK-ir terminals formed symmetric, which was a dominant pattern, and asymmetric synaptic connections with GABA-ir and immunonegative cell bodies and dendrites. Conclusion The ENK-ir terminals might take part in the transmission and regulation of the taste information in the rNTS through inhibiting, exciting the GABAergic neurons or inhibiting d Related Articles | Metrics

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EFFECT OF STZ INTRACEREBRORENTRICULAR INJECTION ON EXPRESSIONS OF APOPTOSISASSOCIATED PROTEINS IN HIPPOCAMPAL NEURONS OF RATS 2007, 38 (2):  163-167.  doi: Abstract ( )   Objective To investigate the effect of insulin signaling pathway on neuronal survival and the effect of the peptide App17 on regulating the expression of some apoptosisrelated proteins in neurons of the hippocampus through intracerebrorentricular injection of streptozotocin in rats. Methods The rats were injected with App17 peptide subcutaneously three weeks after the model group was established by intracerebrorentricular injection of streptozotocin. After the fourweek treament, the expressions of the apoptosisrelated proteins, such as Bcl-2, Bax, CytoC in neurons of the hippocampus were tested with immunohistochemical staining and Western blotting. Results Bax, CytoC positive neurons were widely distributed in the hippocampus of the model group, and the cytoplasm was darkly stained. In contrast, the positive neurons for Bax, CytoC in hippocampus were poorly stained in the control group and the treated group, and appeared significant difference in cell counting as compared with model group. Bcl-2 positive neurons were widely distributed in the hippocampus in the control group and the treated group, and the cytoplasm was darkly stained while its positive neurons were poorly stained in the model group, and appeared significant difference in cell counting as compared with the model group. From the Western blotting clear bars could be seen in the three groups and there was a significant difference between them.Conclusions The expression of Bax, CytoC increased in the hippocampus in the rats with intracerebrorentricular injection of streptozotocin while the expression of Bcl-2 decreased. The App17 peptide could promote the rehabilitation of the abnormal expression of the three proteins to some extent. The insulin signaling pathway could affect the survival of the neu Related Articles | Metrics

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ISOLATION AND IDENTIFICATION OF CIRCULATING FIBROCYTES FROM HUMAN PERIPHERAL BLOOD 2007, 38 (2):  168-172.  doi: Abstract ( )   Objective To establish an effective method of isolating and culturing circulating fibrocytes from human peripheral blood and study the relationship between the expression specific molecule markers expression and the morphological characteristics. Methods Total peripheral blood leukocytes were isolated from human peripheral blood by being centrifuged over FicollPaque and cultured in DMEM supplemented with 20% fetal calf serum. Adhered cells were detected with immunocytochemistry, FCM and electron microscopy. The collagen synthesis was studied by measurement of the hydroxyproline concentration in the medium with chemical method. Results After 9 days cultue in vitro, CD34,CD45 and collagen Ⅰ staining was positive and 83.5% of these cells secreted collagen Ⅰ detected by FCM. Electron microscopy of circulating fibrocytes showed morphological characteristics of fibroblasts. The hydroxyproline concentration in the medium was 11.17%mg/L, which was statistically and significantly different compared with 8.07mg/L in the control medium(P<0.01).Conclusion Circulating fibrocytes could not only be isolated effectively from human peripher Related Articles | Metrics

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THE CONSTRUCTION AND SIGNIFICANCE OF THE HIGHLY EFFICIENT EUKARYOTIC EXPRESSION VECTOR CARRYING HUMAN PPARδ GENE 2007, 38 (2):  173-177.  doi: Abstract ( )   Objective To construct a highly efficient eukaryotic expression vector carrying human peroxisome proliferatoractivated receptor δ(hPPARδ) gene in order to provide an ideal molecular platform for screening natural ligands and functional study of hPPARδ. Methods hPPARδ gene, cloned from total RNA of HepG2 cells by RT-PCR, was ligated with pIRES2EGFP plasmid which was excised by BamHI and SalI double endonucleases. The recombinant plasmid was transfected into 293 cells. Realtime quantitative PCR and immunocytochemistry assays were used to analyze the expression levels of hPPARδ in the transfected 293 cells. Results hPPARδ gene sequence contained in the recombinant plasmid phPPARδIRES2EGFP was verified correct by enzyme digestion as well as sequence analysis. After being transfected into 293 cells, high efficient expression of hPPARδ gene contained in phPPARδIRES2EGFP plasmid was found to display a highly efficient expression detected both at mRNA and protein levels by r Related Articles | Metrics

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THE EFFECTS OF DIFFERENT INDUCERS ON THE DIFFERENTIATION OF NONHEMATOPOIETIC STEM CELLS INTO NERVE CELLS IN HUMAN UMBILICAL CORD BLOOD (HUCB) 2007, 38 (2):  178-181.  doi: Abstract ( )   Objective To explore the best condition for nonhematopoietic stem cells in human umbilical cord blood(HUCB) to differentiate into nerve cells. Methods The mononuclear cell fraction was obtained by Ficoll gradient separation. Immunophenotyping of HUCBderived nonhematopoietic stem cells was detected by flow cytometry. The HUCB derived nonhematopoietic stem cells were stained by immunohistochemical staining after 4 days of teatment with RA, EGF+bFGF, RA+EGF+bFGF and DMSO+BHA. Results The HUCBderived nonhematopoietic stem cells were CD90,CD166 and HLA-ABC positive, but CD34 negative and expressed the neural markers β-tubulin-Ⅲ(neurons), GFAP(astrocytes), and GalC(oligodendrocytes)after being treated with different inducers. Interestingly, the cells showed a high commitment to oligodendrocytes(57.02%), the positive cell numbers of Gal-C>β-tubulin-Ⅲ>GFAP.Conclusion RA is the best Related Articles | Metrics

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INHIBITION EFFECTS OF VARIOUS GASTRINshRNAs ON GASTRIN EXPRESSION IN GASTRIC CANCER CELL LINE BGC-823 2007, 38 (2):  182-186.  doi: Abstract ( )   Objective To study the inhibition effects of various gastrin shNAs on gastrin expression in gastric cancer cell line BGC-823. Methods Four nucleotide sequences of shRNA were designed corresponding to various sites of gastrin gene. Four shRNAs were synthesized by in vitro transcription and transfected into gastric cancer cell line BGC-823 at the final concentration of 10nmol/L,20nmol/L,40nmol/L and 80nmol/L respectively.In situ hybridization and immunohistochemistry techniques were applied to investigate the inhibition of gastrin expression and screen the most effective shRNA. The inhibitory effect on gastrin mRNA of screened shRNA was further identified by RT-PCR. MTT assay was used to determine the inhibitory effect of 4 shRNAs at various final concentrations on the growth of BGC-823 cells. BR> Results The gastrin mRNA and protein exression were suppressed distinctly 24,48,and 72hours after transfection, and exhibited timeand concentrationdependent tendency. The highest suppression efficiency on both mRNA(54.27±0.042)% and protein(41.69±0.038)% level occurred 72 hours later in the cells transfected with shRNAs. The RT-PCR result showed that the inhibitory ratio of shRNA3 on gastrin mRNA of BGC-823 was 481%. MTT displayed a proliferative inhibition of the BGC-823 cells after transfection of shRNAs with a concentrationdenpendent tendency except the shRNA4 treated cells.Conclusion Four gastrinshRNAs showed a significant inhibition effect on gastrin expression of gastric cancer cell BGC-823 on mRNA and protein level. shRNAs might be the most effective Related Articles | Metrics

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AGING EFFECT ON THE THICKNESS OF THE WHOLE RETINA AND ITS SUBLAYERS：A STEREOLOGY STUDY 2007, 38 (2):  187-191.  doi: Abstract ( )   Objective To compare the thickness of the retina and its sublayers between young and elderly rats using a stereological method. Methods Six young(3 months old) and six elderly(2 year old) Long-Evans rats were used in this study. The right eyeball was dissected from each rat and prepared as a set of serial sagittal sections and applied with HE staining. The sections and fields were sampled in the systematic random fashion and examined under a light microscope. The thickness of the whole retina and its 8 sublayers were identified and measured. Results Compared with that of the young rats, the thickness of the whole retina and most of the sublayers of the elderly rats were significantly decreased. The decrease was such so that the proportion of the thickness of each sublayer to that of the whole retina remained unchanged. Most interestingly, among the 8 sublayers of the retina, the thickness of the exterior plexus layer reduced nearly 462% during aging process.Conclusion Aging has a significant effect on the thickness of the rat retina. Such effect is better presented with the systematic random sampling method Related Articles | Metrics

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THE DIFFERENTIATION OF HUMAN UMBILICAL CORD BLOOD CD34SUP>+/SUP> CELL INTO INSULINSECRETING CELLS INDUCED BY NICOTINAMIDE, BETACELLULIN, BFGF AND HGF 2007, 38 (2):  192-195.  doi: Abstract ( )   Objective To investigate the differentiation of CD34SUP>+/SUP> cells derived from human umbilical cord blood into insulin secreating cells in an in vitro system supplemented with growth factors. Methods CD34+ cells were isolated by magnetic cell sorting(MACS). The purity of acquired cells was determined by FACS analysis. Purified CD34+ cells were cultured in DMEM supplemented with 5% FBS, 1×ITS, 10SUP>-4/SUP>mol/L ascorbic acid 2-phosphate, 4.7mg/L linoleic acid, and a combination of nicotinamide, betacellulin、bFGF and HGF. Cultured cells were collected on the 14th and 24th days after the induction to determine the extent of the cell conversion by microscopic observation on morphology, RT-PCR on the transcription of nestin, ngn3 and IPF-1 mRNA, immunocytochemistry on the expression of nestin and insulin, ELISA on the quantification of the insulin product in the medium. Results The isolation purity degree of CD34+ cells was greater than 90% after MACS. Nestin, ngn3 and IPF-1 mRNA were detected in the differentiated cells. Nestin and insulin positive cells were ovserved in the differentiated cells with immumocytochemical staining. The positive ratio of the differentiated cells were (9.8±2.7)%. Insulin ELISA result showed that the insulin product in the culture medium was significantly increased after induction in comparison with that in the control groups (P<0.01).Conclusion The differentiation of CD34+ cells derived from human umbilical cord blood into insulinsecreting cells may be induced by the growth factor mentioned aboveObjective To investigate the differentiation of CD34+ cells derived from human umbilical cord blood into insulin secreating cells in an in vitro system supplemented with growth factors. Methods CD34+ cells were isolated by magnetic cell sorting(MACS). The purity of acquired cells was determined by FACS analysis. Purified CD34+ cells were cultured in DMEM supplemented with 5% FBS, 1×ITS, 10SUP>-4/SUP>mol/L ascorbic acid 2-phosphate, 4.7mg/L linoleic acid, and a combination of nicotinamide, betacellulin、bFGF and HGF. Cultured cells were collected on the 14th and 24th days after the induction to determine the extent of the cell conversion by microscopic observation on morphology, RT-PCR on the transcription of nestin, ngn3 and IPF-1 mRNA, immunocytochemistry on the expression of nestin and insulin, ELISA on the quantification of the insulin product in the medium. Results The isolation purity degree of CD34+ cells was greater than 90% after MACS. Nestin, ngn3 and IPF-1 mRNA were detected in the differentiated cells. Nestin and insulin positive cells were ovserved in the differentiated cells with immumocytochemical staining. The positive ratio of the differentiated cells were (9.8±2.7)%. Insulin ELISA result showed that the insulin product in the culture medium was significantly increased after induction in comparison with that in the control groups (P<0.01).Conclusion The differentiation of CD34+ cells derived from human umbi Related Articles | Metrics

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EFFECTS OF RNAi-MEDIATED GENE SILENCING OF E-CADHERIN EXPRESSION ON BR> THE BIOLOGIC BEHAVIORS OF HO-8910 CELLS BR> 2007, 38 (2):  196-199.  doi: Abstract ( )   Objective In many types of epithelial tumors, downregulation or mutation of the epithelial celladherent molecule E-cadherin is associated with an increased invasiveness that can be prevented by the forced expression of the celladherent molecule. This suggests that E-cadherin is a latestage tumor suppressor that prevents invasion and metastasis. This study was to investigate cell invasion and migration status of human ovary serous cystadeno carcinoma HO-8910 cell line when the E-cadherin expression was downregulated with RNA interference (RNAi) technology. Methods E-cadherin siRNA was transfected into HO-8910 cells to inhibit the expression of E-cadherin. The effect of RNAi was detected by immunofluoresence assay and Western blotting. The invasive ability of the cancer cells was determined by Transwell assay. Results After RNAi, the expressions of E-cadherin were significantly decreased from 63.7% to 11.9% (P<0.01 ), which indicated that RNAi was effective. The cells invasion abilitiy and migration abilitiy were significantly increased. The cells that invaded through the basement membrane filter were increased from 13.4±3.93 to 42.04±4.15 (P<0.01) while the cells that migrated through the basement membrane filter were increased from 23.24±3.71 to 82.0±5.51 (P<0.01).Conclu Related Articles | Metrics

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THE IMMUNOHISTOCHEMICAL OBSERVATION OF DENDRITIC CELLS AND MACROPHAGES IN THE SPLEENS OF SARS PATIENTS 2007, 38 (2):  200-204.  doi: Abstract ( )   Objective To analyse the number of dendritic cells and macrophages and the size of macrophages in the spleen of SARS patients so as to provide evident for the study of pathology and pathogenesis of SARS. Methods Immunohistochemical method with four specific antibodies(S100, CD68, HLA-DR, CD83) were used to detect the dendritic cells and macrophages in the spleens of six dead patients of SARS and six accidental deaths as the controls, The number or the size of these positive cells was analysed with image analysis system. Results In the spleens of SARS patients, the number of S100+ dendritic cells in the white pulp was reduced by 80.4% on average, and even disappeard. The number of CD68+ macrophages in the red pulp was reduced by 39.48% in SARS spleens, and the average size of individual macrophages was increased by 2.21 times. HLA-DR\++ antigen presenting cells (APC) reduced remarkably in the SARS spleen white pulp. CD83+ mature dendritic cells did not exist in either SARS spleens or the control spleens.Conclusion The function of antigen presentation had been damaged severely, which supports that SARS should be categorized as a viral immune deficiency disease. SARS virus doesn’t induce the maturation of DC.The increase in the size of macrophages indicated that they were in an activated state and may play a role in the pathogenesis of SARS. Related Articles | Metrics

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THE EFFECTS OF FSH ON THE PHOSPHORYLATION OF SMAD2/SMAD3 PROTEIN IN RAT OVARIAN GRANULOSA CELLS 2007, 38 (2):  205-208.  doi: Abstract ( )   Objective To study the effects of FSH on the phosphorylation of Smad2/Smad3 protein in rat ovarian granulosa cells. Methods The granulosa cells of 21-day-old SD female rats were cultured with TGFβRⅡ antibody and FSH of different concentrations,and then the cells were obtained at defined points to determine the phosphorylation of Smad2/Smad3 by immunocytochemistry. Results 1. Both Smad2 and Smad3 proteins were mainly expressed in the cytoplasm of ovarian granulosa cells,and a small amount of phosphorylatedSmad2/3 were expressed in the nucleus; 2.The treatment with FSH increased the nuclear transfer of Smad2/Smad3 and the expression of P-Smad2/P-Smad3 in granulosa cells,The effects of FSH were time and dose dependent; 3.The antibody-neutlised TGFβRⅡ inhibited the stimulation of FSH on Smad2/3 in granulosa cells. Conclusion 1.FSH stimulates the phosphorylation of Smad2/Smad3; Related Articles | Metrics

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P>THE INFLUENTIAL FACTORS AND OPTIMUM CONDITIONS OF ELECTRIC TRANSFECTION ON DENDRITIC CELLS/P> 2007, 38 (2):  209-212.  doi: Abstract ( )   Objective To investigate the method and optimum conditions of electric transfection, and the major influential factors of electransfection efficiency and the survival rate of dendritic cells. Methods RNA was extracted from human hepatocarcinoma cell line (Bel 7402). Purified monocytes as precursor DCs (pDCs) were separated from human peripheral blood cells (PBMCs) by density gradient centrifugalization with lymphocyte gradation fluid and adherence method, pDCs were incubated in RPMI-1640 medium containing rhGM-CSF (8×10SUP>5/SUP>IU/L) and rIL-4 (5×10SUP>5/SUP>IU/L) for 7 days and made them fully differentiate into immature DCs(imDCs). The total RNA human hepatocarcinoma cell and green fluorescent protein (GFP) were electransfected respectively into imDCs by electroporation apparatus with different electric voltages, times of impulse, cell concentrations, temperatures and electroporation buffers. Numbers of green fluorescence positive cells and the total cell number were counted respectively under fluorescent microscope, and visible light microscope. One day after the electric transfection, the cells were stained with 0.4% trypan blue, and electransfection efficiency and the cell survival rate were counted. Results Electransfection efficiency was increased to the highest value, up to about 49.7% when imDCs with the concentration of 5×10SUP>6 /SUP>cells/ml were mixed with 40μg-total RNA of human hepatocarcinoma cell, the electric voltage of electroporation apparatus was set at 300V, and the time of impulse was 500Us. Conclusion Electric transfection provides a technical possibility to make human hepatocarcinoma RNA into imDCs. The major influential factors of the electransfection efficiency were electric voltage and impulsing time. As receptor cells, the imDCs growing condition was also an Related Articles | Metrics

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THE HISTOLOGICAL OBSERVATION OF THE STEPWISE DEVELOPMENT OF NEONATAL MOUSE TESTIS IN IMMUNODEFICIENT MICE 2007, 38 (2):  213-217.  doi: Abstract ( )   Objective To investigate the development of neonatal mouse testis in castrated immunodeficient mice by monitoring the graft survival and weight and observing the structure of seminiferous epithelium and the composition of spermatogenic cells in grafts. Methods Neonatal Kun ming mouse testis were grafted under the skin of castrated nude mice (7-12week-old). Grafts were then taken out at different time intervals (namely 16 time points: 3 days, 1-11 weeks respectively and 3-6 months respectively). The survival rate of grafts was calculated and the wet weight was measured. Histological analysis was performed for the observation of the structure of seminiferous epithelium and the composition of spermatogenic cells in grafts. Results Four hundrcd and five grafts recovered out of 450 testis grafted, resulting in a recovery rate of 90.0%. The graft weight increased more than 40 times. The developmental pattern of seminiferous tubules and the appearance time of various spermatogenic cells in grafts were similar as seen in intact mice. Eight weeks after the grafrting, an increasing degradation of seminiferous epithelium was observed.Conclusion When neonatal mouse testis were grafted into nude mice, the developmental course was similar as that in normal donors. The optimal retrieval time of round spermatids and sperms was around the end of the first spermatogenesis wave, about 5-7weeks after the grafting procedure. Related Articles | Metrics

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THE EFFECTS OF HYPERGLYCEMIA ON PREOVULATORY OOCYTE MATURATION，DEVELOPMENT AND GRANULOSA CELL APOPTOSIS 2007, 38 (2):  218-221.  doi: Abstract ( )   Objective To study the effects of hyperglycemia on preovulatory oocyte maturation, development and granulosa cell apoptosis. Methods Female mice (20-day-old) were used to generate diabetic models. Diabetic group and agematched controls received superovulation with 10 IU of PMSG and hCG. At the appropriate time points following hCG injection (t=0h, 2h, 6h and 10h), the ovaries were removed and then paraffin embedded. The expression of connexin-43 was detected by immunohistochemistry. The apoptosis of granulosa cells observed was by TUNEL staining. Results 1.Both the ovaries’ wet weight ratio to the body weight and the number of eggs ovulated were lower in the hyperglycemia group than those in the control group. 2.The expression of connexin-43 detected by immunohistochemistry in the follicles in the hyperglycemia group was greater than that in the control group at the 6th and 10th hours after hCG injection. 3.There were remarkably more TUNELpositive follicles in the diabetic follicles than in the nondiabetic group. And the difference was the most notable at the 6th and 10th hours after hCG injection. 4.Lower percentage of oocytes reached GVBD in the diabetic than that in the controls at the 6th and 10th hours.Conclusion Delayed maturation was observed in the oocytes from those hyperglycemia mice. Hyperglycemia increased granulosa cell apoptosis. The early developmental delay of oocytes may correlate to the Related Articles | Metrics

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THE STUDY OF THE EFFECTS OF SOYBEAN ISOFLAVONE ON ANTIOXIDATION AND BONE MORPHOLOGY IN OVARIECTOMIZED RATS 2007, 38 (2):  222-225.  doi: Abstract ( )   Objective To study the effects of soybean isoflavone(SI) on antioxidation and bone morphology in ovariectomized rats. Methods Seventy female SpragueDawley(SD) rats were randomly divided into 7 groups, according to the levels of total cholesterol in serum: highlipoids group, estrogen group, low dose SI group, middle dose SI group, high dose SI group, sham group and normal control group. A week after bilateral ovaries were excised except in the sham and normal control groups estrogen, different doses of SI or deionized water were fed with intragastric administration for 12 weeks. Body weights were measured every week and vena caudalis serum, innards tissue, femoral bone were collected immediately after the rats were ovariectomized or killed. The activity of alkaline phosphatase(AKP), superoxide dismutase(SOD), glutathione peroxidase(GSH-Px) in serum and bone density were measured respectively, with 63 SD rats being used for the experiment. Results In the intervention groups femoral bone weights were significantly higher than those in both the highlipoids and the control group. The activity of AKP、GSH-Px was increased after intervention, There was a same efficacy between high dose intervention and estrogen treatment on bone density. SI invention mitigated ovariectomyinduced bone loss.Conclusion The SI has some efficacy in ovariectomized rats antioxidation, activity of bone metabolism enzyme and can reverse bone loss. The decrease in bone density is interrupted under sufficient intervention. In view of phytoestrogen’s weak capability of binding with animals’ estrogen receptor(E Related Articles | Metrics

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CHANGES OF 5-HT AND ITS RECEPTOR, IL-2, IL-6 IN ILEUM DURING EXPERIMENTAL SPLEEN-DEFICIENCY COMBINED WITH GASTRIC ULCER IN RATS 2007, 38 (2):  226-230.  doi: Abstract ( )   Objective To study the changes of 5-hydroxytryptamine(5-HT)，its receptor, IL2 and IL-6 in rats’ ileum with spleen deficiency combined with gastric ulcer. To explore the pathogenesis of this disease and the therapic mechanism of sijunzi decoction. Methods Immunohistochemical technique, image analysis, radioimmunoassay and Western blotting were used. Results The 5-HT-positive cells of rat ileum were detected in mucosal and glandular cells.The 5-HTR-positive cells of rat ileum were found in the connective tissues between the intestinal villus and intestinal glands, and in neuroplex and smooth muscle tissues. The expression of 5HT and 5-HTR in ileum increased during the experimental spleen deficiency combined with gastric ulcer in rats. The 5-HTR level of rat ileum was higher. After treatment with Sijunzi decoction, all the changes have been reversed. The level of IL-2 and IL-6 increased during the early period of this disease. When the disease doveloped, the synthesis or secretion of IL-2 and IL-6 decreased. Conclusion The increased contents of 5-HT and its receptor in ileum tissues might be one of the causes of spleen-deficiency combined with gastric ulcer in rats. The mechanism of Sijunzi decoction is to reverse the changes. And the changed activity of IL-2 and IL-6 may be related with the degree of Related Articles | Metrics

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GANODERMA SPORES REDUCING THE OCCURRENCE OF EMBRYONIC NEURAL TUBE DEFECTS INDUCED BY RETINOIC ACID IN PREGNANT MICE 2007, 38 (2):  231-237.  doi: Abstract ( )   Objective To explore whether ganoderma spores could help the neural epithelial cell of embryonic neural tube stagnated by retinoic acid in G\-0/G\-1 phase reenter the cell cycle, keep on proliferating and differentiating, and could reduce the occurrence of neural tube defects (NTDs). Methods When E7.75d, mice of the control group and the ganoderma spores group were given intragastrically onetime retinoic acid all-trans. Then the ganoderma spores solution was given intragastrically to the ganoderma spores group. At E10.5d, The embryos of the two groups were taken out and the ratio of NTDs was counted respectively Immunofluorescence histochemistry and flow cytometry were applied to examine the expression of nestin, and DNA quantity staining and RT-PCR to detect the mRNA transcriptions of Cdk2 and Cdk4. Results The radio of NTDs in the ganoderma spores group was lower than that in the control group remarkably, but the level of nestin was higher. With the neural tube cell cycles compared the cell radio of G\-0/G\-1 phase in the control group was higher than that in the ganoderma spores group, while the ratio in the S phase in the control group, it was lower than that of the normal embryos in the ganoderma spores group. The neural epithelial cell of embryonic neural tube in the ganoderma spores group could transcript Cdk4 mRNA normally, with a low transcription rate in the control group.Conclusion Ganoderma spores can reduce the occurrence of embryonic NTDs induced by retinoic acid in pregnant mice. Related Articles | Metrics

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IMMUNOHISTOCHEMICAL LOCATION OF CD20 POSITIVE CELLS IN THE EARLY HUMAN EMBRYOS 2007, 38 (2):  238-240.  doi: Abstract ( )   Objective To investigate the distribution and significance of CD20 positive cells in the early human embryo. Methods Distribution and morphology of CD20 positive cells in 10 specimens of human embryo aged from 6 to 7 weeks were brown, and studied with immunohistochemical method. Results 1. CD20 positive cells appeared in the liver of early human embryo.2. The immunohistochemical positive substances were brown, and found chiefly in the nuclei of B cells as unevenly-distributed granules,and were not detected in the cytoplasma and on the cell membrane.Conclusion The accuracy of location and distribution of CD20 positive cells in the nuclei of B cells of early human embryo liver may provide an important clue for further exploration of the functional mechanism of CD20 in the process of B cell proliferation and differentiation. Related Articles | Metrics

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AXON-PROJECTION OF ENDOMORPHIN-2-CONTAINING NEURONS IN THE TRIGEMINAL GANGLION TO THE MEDULLARY DORSAL HORN IN THE RAT 2007, 38 (2):  241-242.  doi: Abstract ( )   Objective To observe whether endomorphin-2-immunoreactive (EM2-ir) neurons in the trigeminal ganglion(TG) project to the medullary dorsal horn(MDH) in the rat. Methods Fluoro-gold(FG) retrograde tracing and immunofluorescent histochemical staining for EM2 were applied. Results After FG was injected into the MDH, many FG labeled neurons with small,medium and large diameters were observed in the TG.There were also aboundant EM2-ir neurons with different diameters in the TG.The majority of FG labeled neurons in the TG showed EM2-immunoreactivities.Almost all of the EM2/FG double-labeled neurons were medium and small in size.Conclusion EM2-ir neurons in the TG project to the MDH in the rat. Related Articles | Metrics

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CHANGES OF OREXIN AND ITS RECEPTORS IN THE RAT HYPOTHALAMUS DURING ESTROUS CYCLE 2007, 38 (2):  243-245.  doi: Abstract ( )   Objective To investigate a possible role of orexin in the regulation of estrous cycle by examining the expression of orexin and orexin receptors(OX1R,OX2R) in the rat hypothalamus during the estrous cycle. Methods The levels of prepro-orexin(prepro-OX),orexin-A,OX1R,and OX2R in the rat hypothalamus during the estrous cycle were evaluated by competitive reverse transcription-polymerase china reaction (RT-PCR) assay. Results Only expression OX1R mRNA during late proestrus was significantly higher than that at metestrus.No significant changes of prepro-OX and OX2R mRNA expression were observed during the various estrous cycle phases.Conclusion Orexin might regulate the secretion of GnRH and/or LH by binding OX1R, contributing to the occurrence of ovulation. Related Articles | Metrics

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ISOLATION, PURIFICATION AND IDENTIFICATION OF SERTOLI CELLS FROM RAT TESTIS 2007, 38 (2):  246-249.  doi: Abstract ( )   Objective To obtain highly pure cultured Sertoli cells from rat testis and identificate cultured cells by in situ hybridization for ABP mRNA. Methods Testes from 18-22 day old SD rats were removed and decapsulated,then chopped and sequentially digested with three enzymes at 37℃: first with 0.25% trypsin for 15 minutes,then with 0.1% hyaluronidase for 30 minutes,at last with 0.1% collagenase V for 2-3 hours.The isolated cells were incubated at 32℃ in a humidified atmosphere of 5% CO2.To increase the purity of the Sertoli cells, the cultured cells were subjected to hypotonic shock(treatment) with 20 mmol Tris-HCl after 48 hours of incubation.After a week’s incubation,the cultured cells were identificed by HE staining,AO fluorescence staining,Feulgen staining,and in situ hybridization with digoxin-labeled rat ABP cDNA. Results Over 95% of the cultured cells were Sertoli cells.Most of the cultured cells expressed ABP mRNA and their structural features were the same as those of the Sertoli cells identified by other methods.Highly pure Setoli cells can be obstained by the sequentiall digestion with three enzymes and hypotonic shock.Conclusion As a specifical secreted protein secreted Sertoli cells in testes,ABP mRNA detected by in situ hybridization is a new, specific,effective identification method of Sertoli cells from testes.Meanwhile as an Related Articles | Metrics

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LYMPHANGIOGENESIS AND ITS SIGNIFICANCE IN PATHOGENESIS AND TREATMENT OF THE RELATED DISEASES 2007, 38 (2):  250-252.  doi: Abstract ( )   Related Articles | Metrics