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2008, Volume 39 Issue 4
06 August 2008 Previous Issue    Next Issue
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DEDIFFERENTIATION OF MACROGLIAL CELLS AFTER OPTIC NERVE INJURY AND ITS INDUCTION WITH PREDEGENERATED COMMON PERONEAL NERVE IN RATS 2008, 39 (4):  447-453.  doi: Abstract ( )   Objective To investigate the dedifferentiation of neuroglial cells and its induction after optic nerve injury. Methods Adult male SD rats were randomly divided into 4 groups the normal control group, the injury group, the transplantation group and the microcrush and transplantation group. Optic nerves were harvested at days 3, 7, 14 and 28 after the operation. HE staining was used to count the number of neuroglial cells. Immunohistochemistry, Western blotting and in situ hybridization histochemistry were employed together with computerized image analysis to evaluate the expressions of Nestin, GFAP, MBP, NF, BDNF, Nogo-A and Nogo-A mRNA. Immunofluorescence double staining was used to detect the co-expression of Nestin and GFAP or Nestin and MBP. Results The number of cells only increased at day 7 after the nerve injury, the expressions of Nestin, MBP, Nogo-A and Nogo-A mRNA were upregulated, the expressions of GFAP, NF and BDNF were down-regulated, and some Nestin GFAP positive cells and a few of Nestin-MBP positive cells were detected in the injury group. Compared with the injury group, the number of cells was increased sometime after the nerve injury; the expressions of Nestin, GFAP, BDNF and NF were up-regulated, the expressions of MBP, Nogo-A and Nogo-A mRNA were down-regulated, and the number of Nestin-GFAP positive cells increased in the transplantation group and the microcrush and transplantation group. Conclusion After optic nerve injury, some astrocytes undergo dedifferentiation while the macroglial cells display a gene expression pattern that is unfavorable for nerve regeneration. Pre-degenerated peripheral nerves could enhance the dedifferentiation of astrocytes and induce the gene expression pattern of macroglial cells that is favorable for nerve regeneration. Related Articles | Metrics

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THE EXPRESSION OF REGENERATION GENE PROTEIN2 IN THE SPINAL CORD TRAVERSE INJURY MODEL 2008, 39 (4):  454-459.  doi: Abstract ( )   Objective To investigate the expression of regeneration gene protein2 (Reg-2) after the transection injury in rat spinal cord. Methods Forty five SD rats were randomly divided into two groups: in the injury group, the SCI was produced by complete transection of the cord at the 9th thoracic level, and the sham operation rats were taken as the normal control. The animals were perfused at 1h,1d,3d,7d,14d after the operation, and the spinal cord was taken out at different time points. The expressions of Reg-2 were tested by immunohistochemical analysis and Western blotting, the double staining of Reg-2 and different neural cells special markers(GFAP, Olig2，NPY，CGRP， GAP-43, et al)was done with immunofluorescence method. Results The expression of Reg-2 was found at 1h after the spinal cord injury, reached the peak on day 3 after the injury in the neurons of dorsal horn and day 7 in the neurons of ventral horn. The high expression persisted for 1 week, then decreased gradually.Conclusion Reg-2 may work as an important growth factor and participate in the regeneration and reha Related Articles | Metrics

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GLIAL ACTIVATION AND PROINFLAMMATORY CYTOKINE EXPRESSION IN THE PERIAQUEDUCTAL GRAY INDUCED BY PERIPHERAL INFLAMMATORY PAIN 2008, 39 (4):  460-465.  doi: Abstract ( )   Objective To observe the activation of astrocytes, microglia and expression of proinflammatory cytokines in the periaqueductal gray (PAG) by Complete Freunds Adjuvant (CFA)induced inflammatory pain. Methods Heat hyperalgesia and mechanical allodynia were observed and the expressions of mRNA, protein of glial markers， and proinflammatory cytokines in the PAG were examined following intraplantar administration CFA in right hindpaw of the rat by behavioral testing, reverse transcription polymerase chain reaction (RT-PCR ), Western blotting, and immunohistochemistry. Results CFA injection induced chronic thermal hyperalgesia and mechanical allodynia. After injection, the thermal hyperalgesia was recovered on Day 14, but the mechanical allodynia lasted more than 21 days. Microglial marker (Cluster of differentiation 14, CD14) and proinflammatory cytokines (Interleukinin-1β, tumor necrosis factorα) showed a significant increase in their expression during all phases (acute, subacute and chronic) of inflammation. GFAP was observed only in PAG at subacute and chronic phases of inflammation. Conclusion The activation of microglia and astrocytes may be involved in the initiation and maintenance of inflammatory pain. Enhanced cytokines expression might play Related Articles | Metrics

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THE EXPRESSION OF Wnt SIGNALING GENE FOLLOWING SPINAL CORD COMPLETE TRANSECTION INJURY 2008, 39 (4):  466-469.  doi: Abstract ( )   Objective To study the changes in the expressions of Wnt signaling gene in spinal cord at different times following spinal cord complete transection injury in rats. Methods Thirty healthy Wistar rats were randomly divided into 2 groups:the sham group (only laminectomy, EM>n/EM>=5) and the spinal cord injury (SCI) group (EM>n/EM>=25). The T11 spinal cord segment of adult rats was transected completely in the SCI group. The locomotor functional recovery of hind limbs was observed by grading the BassoBeattieBresnahan(BBB) scale. By using semiquantitative RT-PCR method, the expression of Wnt signaling gene in the injured tissues of spinal cord was detected. Results The hind limbs of rats were completely paraplyzed in the SCI group and their BBB scales were significantly lower than those in the sham group. In the SCI group, the expression of Wnt3a gene had a remarkable increase both on the rostral and the caudal sides at postoperative days 1, 3, 7; on the caudal side, it was higher than that on the rostral side at the same time and had a second increase at day 21 after the operation. The expression of Ngn1 was not detected. Hes1 mRNA was significantly decreased at day 1 after the operation, and increased at day 3 and then gradually decreased. Conclusion Wnt signaling may play an important role in the neural regeneratio Related Articles | Metrics

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NEUROAPOPTOSIS OF CEREBELLAR CORTEX DURING THE DEVELOPMENT IN MOUSE 2008, 39 (4):  470-474.  doi: Abstract ( )   Objective To investigate the rules and mechanism of neuroapoptosis during the development in mouse cerebellar cortex. Methods The neuroapoptosis of cerebellar cortex was visualized by the active Caspase-3 pAb immunohistochemistry and Hoechst 33258 staining from postnatal day 0 (P0) to adult mice. Westernblotting was used to study the expression of Caspase-3 and Caspase-8 in the cerebellum. Results Apoptotic cells density was the highest at P8，P5 and P9 in the external granular layer (EGL)，Purkinje cell layer (PCL) and internal gran BR>ular layer (IGL) respectively, and the density of apoptotic cells of each layer was lower in cerebellar cortex at P20. Activated Caspase-3 was expressed higher at P5, then decreased gradually until the disappearance at P14, at the same time activated Caspase-8 was expressed higher from P0 to P10, and then disappeared at P30. Conclusion It was an important period for neuroapoptosis in cerebellar cortex from P0 to P14, and cell apoptosis pathway that Caspase-3 was activated through cleaved Caspase-8 exists during the molding and growth in the mouse cerebellar Related Articles | Metrics

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CHANGES OF LEARNING， MEMORY AND LEVELS OF CAMKⅡ, CAMmRNA, CREBmRNA IN THE HIPPOCAMPUS OF CHRONIC MULTIPLESTRESSED RATS 2008, 39 (4):  475-480.  doi: Abstract ( )   Objective To investigate the effects of chronic multiplestress on learning and memory and on the levels of calcium/calmodulindependent protein kinase Ⅱ (CaMKⅡ), calmodulin (CaM) mRNA, and cAMPresponse element binding protein (CREB) mRNA in the hippocampus of rats and possible mechanisms. Methods The Wistar rats were divided randomly into stressed (n=16) and control groups (EM>n/EM>=16).The stressed group was given chronic multiplestress for 6 weeks to prepare the chronic multiplestressed model. The rats’ performance of spatial learning and memory was tested by Morris water maze (MWM) and Ymaze. The expressions of CaMKⅡ, CaM mRNA and CREB mRNA in hippocampus of rats’ were detected by immunohistochemistry, Western blotting and RT-PCR respectively. The width of synaptic cleft and the thickness of post-synaptic densities (PSD) were observed in the hippocampal CA3 region of rats under electron microscopy. Results After the exposure to chronic multiplestress for 6 weeks, the ability of learning and memory of the stressed group was higher than that of the control group (P<0.05, P<0.01). The width of synaptic cleft was 5.0580±1.4216 and the thickness of PSD was 20.9547±2.5693 in the hippocampal CA3 of stressed rats. Compared with the control group, the CaMKⅡ immunostaining of stressed animals was stronger in the stratum radiatum and oriens of hippocampal CA1 and CA3, especially in the stratum oriens. The expressions of CaMKⅡ, CaM mRNA, and CREB mRNA in the hippocampus of the stressed group was significantly higher (P& Related Articles | Metrics

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THE INFLUENCE OF INTRANIGRAL INJECTION OF LIPOPOLYSACCHARIDE ON THE MOTOR BEHAVIOR AND DOPAMINERGIC NEURONS IN THE MI>onKEY/I> 2008, 39 (4):  481-487.  doi: Abstract ( )   Objective To observe the effects of intranigral injection of lipopolysaccharide (LPS) on the motor behavior and dopaminergic neurons (DA) in the brain of rhesus mI>onkey/I>, in order to explore the mechanism of inflammation in the progressive degeneration of the dopaminergic neurons. Methods Five adult rhesus mI>onkey/I>s（Macaca, M1-M5, 4 males and 1female）were taken for the experiments. Among them 4 mI>onkey/I>s were intranigrally injected with LPS and survived 24 weeks (M1and M2) and 48 weeks (M3 and M4) respectively after the operation. One mI>onkey/I> (M5) was only injected with saline as a control animal. The injections (LPS or saline) were stereotaxically made 3 times (at weeks 0, 8, 16 respectively; 40μl for each) in the substantia nigra on one side. Moving distance and speed were measured by Ethovison software . The movement time of upper limbs was recorded by using a MI>onkey/I> Automated Movement Assessment Panel. The number of tyrosine hydroxylase (TH)positive neurons and microglia in the substantia nigra was demonstrated by immunohistochemistry staining. Results 1.The tests of locomotion distance and speed in the mI>onkey/I>s decreased apparently after the injection of LPS (24 and 48 weeks) as compared with that in the control animal. 2.The movement time of the upper limb opposed to the injection side decreased also apparently: For M1 and M2, the arm movements were prolonged by 36.1% and 27.1%; the hand movements were prolonged by 41.2% and 28.6%; for M3 and M4, the arm movements were prolonged by 76.5%(P<0.01) and 50%(P<0.01), and the hand movements were prolonged by 81.8%(P<0.01) and 55.6%(P<0.01). No changes could be seen in the limbs, ipsilateral to the injection, neither in the control case, M5. 3.The number of TH-positive neurons in the injected substantia nigra decreased to some degrees, which was more obvious in the cases of M3 and M4. 4.The microglia were activated in the area in all LPSinjected cases especially in the cases of M1 and M2. Conclusion The injection of LPS can induce brain inflammation which leads to a long-term an Related Articles | Metrics

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USING PHOTOSHOP IN THE THREE DIMENSIONAL RECONSTRUCTION OF CORTICOSPINAL TRACTS IN THE SPINAL CORD OF ADULT SPRAGUEDAWLEY RAT 2008, 39 (4):  488-492.  doi: Abstract ( )   Objective To make three dimensional(3D) reconstruction of corticospinal tracts in rat, using serial sections of rat’s spinal cord and tool software for three dimensional reconstruction. Methods The serial transversal sections of rat’s spinal cord were made and mounted directly, then stained with Luxol fast blue(LFB) and their images were taken. The section images were registered, segmented and changed into grayscale images under the Photoshop circumstance. 3D-DOCTOR software was used to make threshold segmentation and to reconstruct the spinal cord, grey matter and bilateral corticospinal tracts of rat. The reconstructed structures were observed at random angles, dissected and measured on a common desktop. Results The reconstructed spinal cord, grey matter, bilateral corticospinal tracts of rat were solid. In C1 to S4 segments of rat’s spinal cord, the corticospinal tracts lay in the posterior funiculus and were close to the back of intermediate zone, appearing as gradually narrowing columns. Conclusion The corticospinal tracts in rat’s spinal cord can be reconstructed and offer survey data by using serial LFB stained spinal cord sections and computer Related Articles | Metrics

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THE EFFECT OF SELENIUM ON THE ACTH POSITIVE CELLS IN THE PARS DISTALIS OF PITUITARY OF RAT WITH COLON CANCER INDUCED BY AZOXYMETHANE 2008, 39 (4):  493-497.  doi: Abstract ( )   Objective To observe the effect of selenium on the ACTH positive cells in the pars distalis of pituitary of rat with colon mucosa cancer induced by azoxymethane(AOM). Methods Twenty 3 weeks old Sprague Dawley male rats were randomly divided into 4 groups: the normal control group; the experimental control group; the selenium before AOM induced colon mucosa cancer group; the selenium after AOM induced colon mucosa cancer group. To induce rat colon mucosa cancer, 3 weeks old SD male rats received i.p. injections of AOM at a dose of 15mg/kg once a week for two weeks. Selenium (NaSUB>2/SUB>SeOSUB>3/SUB>, 4mg/L) began to be provided in the drinking water before and after AOM administration, and lasted for the whole experiment period. The rat pituitary tissues were excised from the 34 weeks old rats. The morphological changes of the ACTH positive cells in the pars distalis of pituitary were observed using immunohistochemical methods， and then the results were analyzed by image analysis system. Results Aberrant crypt foci (ACF) and aberrant crypt (AC) were showed in the colon mucosa of rats that received two weekly i.p. injections of AOM by light microscopy with methylene blue staining. The ACTH positive cells in the experimental group rat displayed a significant increase compared with those in the normal control group rat (P<0.01) with immunohistochemical methods. There were more positive intensity in the cells with ACTH positive cells in the supplement selenium group than that in the experimental group (P<0.01). Conclusion Selenium can promote the function of the ACTH positive cells in the pars distalis of rat pituitary with colon mucosa cancer ind Related Articles | Metrics

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THE CHANGE OF THE BLOODBRAIN BARRIER OF THE EPILEPSY RATS AND THE PROTECTIVE ROLE OF TOPIMMATE 2008, 39 (4):  498-501.  doi: Abstract ( )   Objective To observe the change of the precentral gyrus bloodbrain barrier ultrastructure in the epilepsy rats induced by coriaria lacton and affected by topimmate, and to study the effect of the bloodbrain barrier on the mechanism of the falling sickness and therapy by topimmate. Method Thirty healthy male Sprague Dawley rats were randomly divided to three groups: the control group, the epilepsy group and the treatment group. The coriaria lacton was injected into the lateral ventricles of the rats in the epilepsy and treatment groups, and the epilepsy animal model was established. Gastric perfusion was performed 1 hour after falling epilepsy to the rats of the treatment group and was repeated one time per day for 7 days. The precentral gyrus was cut 7days later, and the ultrathin sections were made. The change of blood brain barrier ultrastructure was observed under electron microscope. Results The endothelium, basement membrane, pericyte of epilepsy group displayed obvious edema. The electron density reduced, and the edema of the treatment group alleviated. There was signifcant difference between the epilepsy group and the control group（P<0.05）,in the thinkness of endothelium cytoplast, basement membrane and pericytes cytoplast of bloodbrain barrier. So was the case between the treatment group and the epilepsy group（P<0.05）. Conclusion 1.Endothelium, basement membrane and pericytes of bloodbrain barrier of the epilepsy rats induced by coriaria lacton displayed obvious edema, and topimmate weakened th Related Articles | Metrics

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PROTEOMIC ANALYSIS OF CELL DIFFERENTIATION IN THE SUBVENTRICULAR ZONE OF Hes5 KNOCKOUT MICE 2008, 39 (4):  502-507.  doi: Abstract ( )   Objective Hes5 is one of the critical effectors of the Notch pathway and its expression can inhibit neuronal differentiation.Which protein expression is influenced in cell differentiation by Hes5 remains to be determined. Methods Experiments were designed to study changes in protein expression in the subventricular zone(SVZ) of the Hes5 knockout mice at the postnatal day 8 using the new method of proteomic isobaric tags for relative and absolute quantification （iTRAQ）, and confirmed expression of the up-gulated and down-regulated proteins by Western blotting. Results Nineteen proteins showed differences in expression levels in the SVZ of Hes5 knockout mice. When compared to the wild type mice, expressions of 11 proteins in the knockout mice were up-regulated, and the differentially expressed proteins showed ratios of knockout/widtype between 1.20 and 1.32. Expressions of 9 proteins were down-regulated, with their ratios were between 0.77 and 0.83. The up-regulated proteins have been shown to be involved in neurogenesis, cell adhesion, migration and signal transduction. The down-regulated proteins have been mainly shown to be involved in growth inhibition and cytoskeleton. Western blotting analysis further confirmed the expression of proteins from iTRAQ. Conclusion Our findings suggest that Hes5 may influence cell differentiation by regulating expression of some proteins involved in cell growth and migr Related Articles | Metrics

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REGULATING EFFECTS OF THE PI3-K/Akt SIGNALING PATHWAY ON THE DIFFERENTIATION OF MARROWDERIVED CARDIAC STEM CELLS 2008, 39 (4):  508-513.  doi: Abstract ( )   Objective To investigate the regulating effects of PI3-K/Akt signaling pathway BR>on the differentiation of the marrowderived cardiac stem cells (MCSCs) induced with BMP2 toward cardiomyocytes and to determine the possible mechanisms in signal transduction on differentiation of MCSCs toward cardiomyocytes. Methods MCSCs were selected from the bone marrow of SD rats with singlecell cloning culture. The differentiation of MCSCs toward cardiomyocytes was induced with 10 μg/L BMP-2, and changes in p-Akt levels in MCSCs were examined with Western blotting and immunocytochemistry using several time points after induced with the BMP-2. Effect of PI3-K was detected with the PI3-K inhibitor Ly294002 Expression of Nkx2-5、GATA-4 and cTnT、Cx-43 mRNA of MCSCs before and after induction were determined with RT-PCR . After induction with BMP-2, the morphological alterations to the cells were examined under a phase contrast microscope. Results MCSCs express ckit and the early cardiac transcription factors Nkx2-5. Before induction with BMP-2,the p-Akt level in MCSCs was low. After induction with BMP-2, the p-Akt level in MCSCs was increased and was to reach a peak at 20 to 30 min and subsequently decreased at 1 hour. After treatment with the PI3-K inhibitor Ly294002, p-Akt level of the cells was decreased significantly. RT-PCR detection showed that expression of Nkx2-5 and GATA-4 were weak before induction; however expression of Nkx2-5 and GATA-4 was increased at two weeks and conspicuous at four weeks after induction. Expression of cTnT and Cx-43 mRNA was not observed before induction, and was obvious at two weeks and even stronger at four weeks after induction. After treatment with Ly294002, the expression of Nkx2-5, GATA-4, cTnT and Cx-43 mRNA was reduced significantly, although no morphological change to the cells was obvious. Conclusion BMP-2 may induces differentiation of MCSCs toward cardiomyocytes, and the PI3-K/Akt signali Related Articles | Metrics

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EFFECTS OF AUTOPHAGY ON CLEARANCE OF THE APOPTOTIC LYMPHOCYTES BY MACROPHAGES 2008, 39 (4):  514-518.  doi: Abstract ( )   Objective To investigate the morphological characteristics of the autophagic structures in macrophages after phagocytosis of apoptotic lymphocytes and to explore the effects of autophagy on clearance of the apoptotic cells by macrophages. Methods Apoptosis of lymphocytes was induced with cyclophosphamide. The morphological changes of macrophages phagocytizing the apoptotic cells were viewed with a scanning electron microscope. The structural features of the autophagosome precursors, autophagosomes and autophagolysosomes in macrophages were examined with a transmission electron microscope, and the cross-section areas of the autophagic structures were measured with an image analyzer. The autophagosomes of macrophages were labeled with monodansylcaolaverine(MDC) staining and quantitated using laser scanning confocal microscopy. Results Macrophages actively phagocytized the apoptotic lymphocytes, apoptotic nuclei, apoptotic bodies and other cell debris to form heterophagosomes. When compared with the control group, numbers of autophagic cells and autophagosomes in these cells increase in the group of macrophages that engulfed the apoptotic cells. In addition, the ratios of the cross-sectional areas of the autophagic structures to that of the cytoplasm of the macrophages were greater. There were also apoptotic bodies or other cell debris in many the autophagosomes, and these autophagosomes were large and near the cell membrane. Autophagosomes containing the whole apoptotic cell or apoptotic nucleus were not observed. Conclusion The autophagic abilities of macrophages were significantly enhanced when the cells removed the apoptotic lymphocytes. Autophagy also plays an important direct or indirect roles in clearance of the apoptotic lymphocytes by macrophages. Related Articles | Metrics

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RELATIONSHIP BETWEEN EXPRESSIONAL PROFILES OF APOPTOSIS RELATED GENES AND APOPTOSIS ACTION OF THE RAT REGENERATING LIVER 2008, 39 (4):  519-525.  doi: Abstract ( )   Objective To investigate the actions of apoptotic pathways in rat liver regeneration (LR) at the transcription level. Methods The apoptosis genes were obtained by referring to the putative literatures and databases. Apoptoticrelated genes expression profiles in rat LR were checked by Rat Genome 230 2.0 array. Identification of LR-associated genes was made by comparing the discrepancies of genes expression between partial hepatectomied (PH) and sham operatied (SO) rats. Results 252 genes were found to be related to liver regeneration. The initial and total expressing gene numbers occurring in the initiation phase of liver regeneration (0.54hours after PH), GSUB>0/SUB>/GSUB>1/SUB> transition (4-6hours after PH), cell proliferation (6-66hours after PH), cell differentiation and structurefunction reconstruction (72-168hours after PH) were 81,23,155,16 and 161,100,733,192 respectively. This demonstrated that LR-related genes primarily started their expression in the initiation phase, and worked in the different phases. The total times of their up-and down-regulated expression were 795 and 291, demonstrating that expression of most genes was enhanced in LR. Their expression patterns could be classified into 35 types, indicating the expressions of apoptosisrelated genes in LR were diverse and complicated. Conclusion There are fifteen kinds of apoptosis pathways that associate to apoptosis regulat Related Articles | Metrics

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RELAVANCE OF LIVER STEM CELL GROWTH AND DIFFERENTIATION WITH RAT LIVER REGENERATION 2008, 39 (4):  526-530.  doi: Abstract ( )   Objective To study the function of liver stem cells during at liver regeneration(LR) at transcriptional level. Methods The liver stem cell growthand differentiationrelated genes were obtained by data collection and literature review, and the gene expressions in rat regenerating liver were checked by Rat Genome 230 2.0 array. The relativity of the LRassociated genes was identified by comparing the discrepancy of gene expression changes between partial hepatectomy (PH) group and sham operation (SO) group. Results Fifty genes were found to be related with liver regeneration. The initial and total expressing gene numbers occurring in the initiation phase of liver regeneration (0.5-4 hours after PH), GSUB>0/SUB>/GSUB>1/SUB> transition (4-6 hours after PH), cell proliferation (6-66 hours after PH), cell differentiation and structure function reconstruction (72-168 hours after PH) were 24, 10, 21, 2 and 24, 23, 46, 26 respectively, indicating that the related genes were mainly triggered during the initiation phase, and worked at different phases. The total frequencies of their up- and down-regulation expression were 153 and 123 respectively, demonstrating that the expression of the major genes increased, and the minority decreased. Their expression profiles were classified into 6 types, indicating that the activities mentioned above were diverse and complicated during liver regeneration. Conclusion The growth and differentiation of liver stem cells are enhanced during liver r Related Articles | Metrics

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DERIVATION OF ADIPOCYTES AND NEURONLIKE CELLS FROM CHICKEN EMBRYONIC PRIMORDIAL GERM CELLS IN VITRO 2008, 39 (4):  531-538.  doi: Abstract ( )   Objective To explore the capability of chicken embryonic primordial germ cells (EPGCs) to differentiate into adipocytes and neuronlike cells, and to compare the inductive effect of a variety of specific chemical inductive media and their coordination with variant inductive programmer. Methods The chicken EPGCs at stage 19 and 28 were cultured and subcultured to the 4th passage, and then were differentiated into adipocytes and neuronlike cells respectively. EPGCs differentiated cells were then identified by oil redO. EPGCs were similarly induced into neuronlike cells in the medium containing RA, BHA, IBMX and DMSO; the differentiated cells were identified by toluidine blue stain and specific immunohistochemistry. Results About 74%-91% oil redO positive EPGCs were induced into adipocytes after 14-21 days. Programmer 2 showed the best result（P<0.01）. The differentiation rates of EPGCs from stage 19 and 28 were 89% and 91% respectively. The cooperative inductive effect was significantly higher than the single inductive effect of desamethason, insulin and IBMX. However, in the same induction media, the oil redO stained cells showed no significant difference between stage 19 and stage 28（P>0.05）. The specific PPARγ gene cDNA had been proliferatied from induced adipocytes. And 75%85% toluidine blue positive EPGCs were induced into neuronlike cells after 3-7 days. The average differentiation rates were 82% and 83% for inductive media RA and IBMX, which were significantly higher(P<0.01）than the cooperative inductive effect of RA,BHA,DMSO and IBMX. There was no signifi Related Articles | Metrics

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IN VITRO STUDIES OF BONE MARROW MESENCHYMAL STEM CELLS ON HA/TCP BONE GRAFT SCAFFOLD 2008, 39 (4):  539-542.  doi: Abstract ( )   Objective To study the adhesion and proliferation of rabbit bone marrow mesenchymal stem cells(BMSCs) on the HA/TCP bone graft scaffold. Methods BMSCs were harvested from the iliac bone and obtained by using adhesive culture method. BMSCs were induced with osteogenic medium, at the 7th day, the cells were stained by the calcium cobalt method to show the activities of alkaline phosphatase (ALP). At the 10th day, the mineralized nodules of osteoblasts stained with chinalizarin. Otherwise BMSCs were induced with adipogenic medium,and at the 21st day the cells stained with oil red O. We cultured the BMSCs on the surface of a matrix scaffold with osteogenic medium. The morphologic characters were checked by light microscopy, fluorescence microscopy and scanning electronic microscopy, and the proliferation of BMSCs were assayed using the MTT test. Results At the 7th day after osteoblastic induction, ALP was strongly positive, and at the 10th day, mineralized nodules stained with chinalizarin were jacinth. At the 21th day after adipogenic induction, the adipocytes were stained with oil red O. BMSCs grew well around or in pores of the HA/TCP bone graft scaffold and could be seen using both fluorescence microscopy and scanning electron microscopy. The MTT test assay showed that the HA/TCP couldn’t inhibit the proliferation of BMSCs. Conclusion BMSCs have a good biocompatibility with a HA/TCP bone graft scaffold. Related Articles | Metrics

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EFFECTS OF RUNX3 ON CELL TURNOVER IN GASTRIC CANCER AND ITS PROGNOSTIC VALUE 2008, 39 (4):  543-546.  doi: Abstract ( )   Objective To evaluate the relationship between the RUNX3 gene, cell turnover(apoptosis and proliferation) and survival in gastric cancer patients. Methods RUNX3 was examined using immunohistochemical staining. Cell turnover was analyzed by flow cytometry using standard techniques. Survival was evaluated by KaplanMeier survival curves. BR> Results The positiveexpression rate of RUNX3 in tumor tissue from 60 patients with gastric cancer was 46.7%. RUNX3 positiveexpression was correlated with lymph node metastasis and distant metastasis(EM>P/EM><0.05). Normal distribution was found by frequency distribution of AI/PI in gastric carcinoma cells. Tumor cells exhibited a wide range of AI/PI expression from 0.05 to 0.46Cell turnover was also correlated with lymph node metastasis and distant metastasis (P<0.05).It was determined that the expression of RUNX3 and cellturnover rates were associated (EM>r/EM>=0.39，EM>P/EM>0.05). Using KaplanMeier survival curves and theEM> Log-rank/EM> test, there was also a correlation between RUNX3 and survival(EM>P/EM><0.05). Conclusion The RUNX3 gene may be related with tumorigenesis and tumor progression by affecting cell turnover, The detection of RUNX3 may be an impor Related Articles | Metrics

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DEVELOPMENTAL CHARACTERISTICS AND GENE EXPRESSION PATTERNS OF MOUSE EMBRYONIC STEM CELLSDERIVED EMBRYOID BODIES IN VITRO 2008, 39 (4):  547-551.  doi: Abstract ( )   Objective To explore the developmental characteristics and gene expression patterns of mouse embryonic stem cells (mESG)derived embryoid bodies(EBs). Methods The EBs were derived from undifferentiated mESCs by suspending culturs in leukemia inhibitory factor (LIF) free media. The morphological character istics and genes expression related to the sequential stages of embryonic development was detected by morphological observation, immunohistochemistry and RT-PCR over 90 day’s duration. Results The primary round and packed EBs were formed within the first 24 hours of suspension culture, which also comprised many of undifferentiated mES. The simple embryonic bodies formed through 3day of differentiation, and the EBs developed to embryonic cavity-like structures at 9 days. Subsequently, the EBs formed a typical and integral trilaminar structure at 11 days. Early stage EBs always developed to a columnar epithelium with the formation of a central cavity and was also cystic. From the seventh week on, the EBs growth ceased. At fifteen day, the EBs derived from mES expressed a trilaminar protein markers Nestin and cTnT, The expression of other genes associated embryonic development such as Vimentin, Nestin , Flk-1、Gata-1, Transthyretin and α-fetoprotein were detected by RT-PCR . Conclusion EBs can differentiate into the dimensional cell aggregates and initiate mES differentiation procedures during the long periods of culture in vitro. Our results provided a molecular Related Articles | Metrics

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EXPRESSION OF ECTONUCLEOTIDEPYROPHOSPHATASE/PHOSPHODIESTERASE 1 IN HUMAN OVARY AND ITS RELATIONSHIP WITH POLYCYSTIC OVARY SYNDROME 2008, 39 (4):  552-556.  doi: Abstract ( )   P>Objective Insulin resistance is a possible cause of polycystic ovary syndrome (PCOS). It is presumed that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is associated with insulin resistance. The purpose of this study is to explore the expression of ENPP1 in granulosa cells of the ovary and its relationship with PCOS. Methods Twelve aliquots of follicular granulosa cells were isolated from 12 samples of the patients with PCOS and 22 aliquots from 22 samples of the patients without PCOS, respectively．ENPP1 expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR ) and in situ hybridization. The relative expression of ENPP1 mRNA was detected by realtime quantitative polymerase chain reaction (realtime PCR). Results ENPP1 was expressed in granulosa cells of the ovary. ENPP1 expres Related Articles | Metrics

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THE EXPRESSION OF ANTYAPOPTOSIS PROTEIN BAG-1 IN NONSMALL CELL LUNG CANCER AND ITS RELATIONSHIP WITH CELL APOPTOSIS AND EXPRESSION OF BCL-2 2008, 39 (4):  557-561.  doi: Abstract ( )   Objective To study the expression and clinical significance of antyapoptosis protein BAG-1 in nonsmall cell lung cancer（NSCLC）,and its relationship with cell apoptosis and expression of Bcl-2 protein. Methods Immunohisto chemistry streptavidinperoxidase conjugated (SP)method was used to examine the expression of BAG-1protein and Bcl-2 protein, and terminal deoxynucleotidyl transferase mediated UTP nick end labeling (TUNEL) method was used to examine the apoptosis index in 54 cases of NSCLC .The expression of BAG-1 protein in 20 cases of normal bronchus mucosa tissue also be detected as control. Results Express positive rate of BAG-1 protein in NSCLC is 7407%, obviously higher than that in normal bronchus mucosa tissue (positive rate is 5%). In cases of NSCLC ,the expression of BAG-1 protein has not correlation with the age, gender, pathologic classification,but have closed correlation with lymph node metastasis，degree of differentiation,pTNM stage(P<0.05).There is positive correlation betweened BAG-1 protein expression and Bcl-2 protein expression（pearson contingency coefficient is 0.406,P=0.003，P<0.01）.Following the increasing of expression degree of BAG-1protein,the apoptosis index of cancer cell was degraded. The statistically significant difference was found between each groups. Conclusion BAG-1 protein is over expression in NSCLC compare with normal bronchus mucosa tissue,have closed relation with NSCLC genesis and development.There is posi Related Articles | Metrics

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THE SIGNIFICANCE OF THE EXPRESSION OF MITOCHONDRIAL DNA Cyt-b AND VASCULAR ENDOTHELIAL GROWTH FACTOR mRNA IN BREAST CANCER 2008, 39 (4):  562-565.  doi: Abstract ( )   Objective To study the significance of the expression of mitochondrial DNA Cyt-b and vascular endothelial growth factor mRNA in human breast cancer. Methods Reversedtranscription polymerase chain reaction（RT-PCR）was applied to detect the expressions of mtDNA Cyt-b and VEGF in 19 samples of breast cancer tissue and 19 samples of peritumoral tissues. β-actin served as a quantitative standard marker. The different expressions in breast cancer tissues and paracancerous tissues were compared and their relations with clinical pathology parameters were analyzed. Results The expressions of mtDNA Cyt-b and VEGF mRNA in 19 samples of breast cancer tissues were higher than those in paracancerous tissues, with a significant difference between the two groups (P<0.01). The expression of mtDNA Cyt-b was positively correlated with the expression of VEGF mRNA (P<0.01) and the expression of VEGF145 and VEGF189 mRNA (all P<0.05). The expression of VEGF145 and VEGF189 mRNA, which were the isomerides of VEGF, was higher in breast cancer tissues than in paracancerous tissues, with a difference between the two groups (P<0.05). The expression of mtDNA Cyt-b and VEGF mRNA in lymph node metastasis group was higher than that of nonlymph node metastasis group, with a difference between the two groups (P<0.05). The expressions of mtDNA Cyt-b and VEGF mRNA in phase Ⅰof TNM stage were lower than those in phase Ⅱ, with a difference between the two groups (P<0.05). There was a significant differe Related Articles | Metrics

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LUNG HISTOGENESIS AND EXPRESSION OF TGF-β IN BUFO RADDEI STRAUCH 2008, 39 (4):  566-572.  doi: Abstract ( )   Objective To analyze the tissue development rule of the Bufo raddei Strauch lung，to observe the expression of transforming growth factor-β（βSUB>1/SUB>，βSUB>2/SUB>，βSUB>3/SUB>）in developing lung，and to discuss their functions and relationships. Methods The tissue development of the （218） Bufo raddei Strauch lungs was studied by biological microscope technology，and the expression of TGF-β was measured in lung development by immunohistochemistry and images were analyzed by IPP，providing a basis for study of the zoology and developmental biology. Results (1) Lung bud occured in larval 22nd stage from pharynx caudal part. In 26th stage the minutenss locule began to appear. The lung bud gave rise to obvious lung cavity in 31st stage. In 33rd stage originality alveolus began to appear. In 36th stage obvious septum and alveolus appeared. There were many capillary vessel in the wall of lung. In 39th stage the lung of tadpole was like as one of young toad. The respiratory epithelium differentiated from columnar into squamous. The entire developmental process was divided into four phases，lung bud Ⅰphase，lung bud II phase，originality alveolus phase and alveolus phase. (2)In the early Related Articles | Metrics

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OBSERVATION OF HUMAN PERIODONTAL LIGAMENT CELLS CULTURED ON ELECTROSPUN PLLA/HA BIOMATERIAL 2008, 39 (4):  573-577.  doi: Abstract ( )   Objective To observe the attachment and growth of human periodontal ligament cells (hPDLCs) on electrospun poly (L-lactic acid)/hydroxyapatite (PLLA/HA) fibrous biomaterial (called "biomaterial" for short), and to investigate the feasibility of applying this new material in human periodontal tissue regeneration. Methods PLLA/HA fibrous biomaterial was fabricated by electrospinning technique. the hPDLCs were harvested from healthy premolars extracted for or BR>thodontics, and the cells were identified by immunohistochemical method. The compatibility of the biomaterial was evaluated by the MTT assay method. the hPDLCs were seeded onto the PLA/HA fibrous biomaterial, and then the compound was observed under scanning electron microscope; the hPDLCs transfected by the recombinant adenovirus containing green fluorescent protein was cocultured with the biomaterial and observed under confocal laser scanning microscope. Results The hPDLCs were successfully separated, and immunohistochemistry staining of vimentin protein showed that the cells came from the mesoderm; the MTT assay results showed that the cells propagated on the biomaterial well compared with cultured normally; the scanning electon microscope (SEM) results showed the cells on the biomaterial grew and stretched well; the confocal laser scanning microscope results showed that the cells grew along the fiber of the biomaterial, and presented a certain three dimensional structure. Conclusion The results suggested that electrospun PLLA/HA fibrous biomaterial possesses a three dimensional net-like structure and a good cellular compatibility with hPDLCs; the hPDLCs can adhere and propagate well on it. This experiment can provide some basic theory for the use of electrospun PLLA/HA fibrous biomaterial in the regeneration of human periodontal tissue. Related Articles | Metrics

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EXPRESSION OF Polo-LIKE KINASE 1 DURING THE DIFFERENTIATION OF CARDIOMYOCYTES IN RATS 2008, 39 (4):  578-581.  doi: Abstract ( )   Objective To understand the mechanism of how cardiomyocytes exit from the cell cycle, we examined the expression of pololike kinase 1(plk1) in the postnatal developmental process of cardiac myocytes. Methods Mitotic Index(MI) of cardiomyocytes was examined in the neonatal, 2weekold, 4weekold, and adult rat hearts (five cases per groups) by double immunofluorescence stained with H3P and α-sarcomeric actin antibodies. plk1 mRNA and protein expression during the postnatal developmental process of cardiac myocytes were detected by RT-PCR and Western blot analysis in rat hearts. Results The MI of cardiomyocytes in 0dayold hearts (0.905±0.087%) was approximately 24 times over that in 2weekold hearts(0.372±0.094%)（P<0.01）. The mRNA expression of plk1 is obviously declined during postnatal 7 days to 10 days in rat hearts（P<0.01）, and was undetectable in the adult group. Related Articles | Metrics

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BIOCOMPATIBILITY OF ATELOCOLLAGEN SCAFFOLD IN THE THREE DIMENSIONAL CULTURED RAT CARDIAC MYOCYTE 2008, 39 (4):  582-585.  doi: Abstract ( )   Objective To investigate the biocompatibility between the atelocollagen scaffold and 3D-cultured myocardial cells，and to find out the condition and an optimal biomaterial scaffold that can be applied to 3D-culture myocardial tissue. Methods The primary cultured myocardial cells were purified and then inoculated into the atelocollagen scaffold. The cells in the atelocollagen scaffold were observed by light microscope, scanning electron microscope(SEM) and transmission electron microscope (TEM) at different times (8d, 16d, 20d). Results On the first day, cardiac cells pulsating together with the atelocollagen scaffold could be detected under the microscope,which were pulsating complex. A plenty of cells in the atelocollagen mesh were observed under the light microscope, and the cells coalesced with the scaffold. The cells were compacted to the scaffold and coalesced with it at three stages under TEM. Those cells were sticked to the atelocollagen scaffold and expanded sufficiently under SEM. On the atelocollagen scaffold surface, these cells coalesced with lamellar.Conclusion The biocompatibility of the atelocollagen scaffold is better for cultured cardiac myocyte. It can be used as a natural material for 3D-cultured cells, and is suitable for 3D-cultured cardiac cells and cardiac tissues. Related Articles | Metrics

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SUPPLY OF PULMONARY BLOOD IN YOUNG CHILDREN WITH TOF AND PULMONARY ATRESIA 2008, 39 (4):  586-589.  doi: Abstract ( )   Objective To analyze pulmonary blood supply in congenital heart disease with tetralogy of Fallot (TOF) and pulmonary atresia in young children. Methods The clinic and imaging data of 94 cases of congenital heart disease with tetralogy of Fallot (TOF) and pulmonary atresia were analyzed, aged 2-26 (11.25±8.24) months, from January 2004 to January 2007 Results There were 51 cases with pulmonary blood flow only from ventricle, 27 cases with pulmonary blood flow only from patent ductus arteriosus (PDA) and/or aortopulmary collateral arteries (APCAs), and 16 cases with pulmonary blood flow from both pulmonary and systemic arterier. There were no significant differences in the incidence of PDA and the degree of pulmonary stenosis (P>005). The differences were significant between the incidence of APCAs and the degree of pulmonary stenosis (P<0.05). Conclusion The source of pulmonary blood in congenital heart disease with TOF and pulmonary atresia is complex. The incidence of aortopulmonar Related Articles | Metrics

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EXPRESSIONS OF Bcl-2 AND Bax PROTEINS IN CHORIONIC VILLUS AND DECIDUAL TISSUE OF HUMAN PLACENTA DURING EARLY PREGNANCY 2008, 39 (4):  590-593.  doi: Abstract ( )   Objective To study the expressions of Bcl-2 and Bax proteins in human chorionic villus and decidual tissue during early pregnancy. Methods The expressions of Bcl-2 and Bax proteins in the cells of human chorionic villus and decidua were observed by immunohistochemistry(ABC) from the 5th to the 7th week in early pregnancy. Integral absorbances (IEM>A/EM>) of Bcl-2 and Bax proteins were detected by using special image analysis software. Results Bcl-2 protein expressed in all syncytiotrophoblast cells and decidual cells, and its protein integral absorbance decreased with time. The difference between any two groups was not significant statistically (P>0.05). From the fifth week to seventh week，no expression of Bax protein observed in syncytiotrophoblast and cytotrophoblastic cells. Bax protein expressed in a few the decidual cells in the sixth week of early pregnancy.Conclusion Bcl-2 and Bax proteins express in early pregnancy, which take part in the multiplication and differentiation of trophoblast cells, and womb decidualization during early pregnancy．They play an important role in the development and morphogenesis of villus, the rebuilding of tissue structure and th Related Articles | Metrics

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EXPRESSION AND SIGNIFICANCE OF nNOS, PAX3 AND CX43 PROTEINS IN EARLY DEVELOPING SPINAL CORD OF HUMAN EMBRYOS 2008, 39 (4):  594-597.  doi: Abstract ( )   Objective To explore the rule of distribution and the expression significance of neuronal nitric oxide synthase (nNOS), paired box 3(Pax3) and connexin 43(Cx43) proteins in the early developing spinal cord of human embryos. Methods During the 5th to 16th weeks of human gestation, the expression productions of nNOS, Pax3 and Cx43 proteins were investigated in the anterior horn of spinal cord using the immunohistochemical methods. Results During the 5th to 16th weeks of human gestation, the immunohistochemical staining intensities of both nNOS and Pax3 in the anterior horn neurons of spinal cord increased from weakly to strongly positive staining. During the 5th to 12th weeks of human gestation, the immunohistochemical negative staining of Cx43 protein was observed in the anterior horn neurons of spinal cord, while the positive staining was observed from the 13th to 16th weeks.Conclusion nNOS, Pax3 and Cx43 proteins are related to the growth and development of spinal cord in human embryos. Related Articles | Metrics

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BIOCOMPATIBILITY OF HUMAN ACELLULAR AMNIOTIC MEMBRANE WITH CULTURED VASCULAR SMOOTH MUSCLE CELLS IN VITRO 2008, 39 (4):  598-601.  doi: Abstract ( )   P>Objective To investigate the biocompatilility of human acellular amniotic membrane (HAM) with vascular smooth muscle cells (VSMCs) and to explore the possibility to construct tissue engineering bladder with HAM as the scaffold and VSMCs as the seed cells. Methods After physical and 1% trypsogen preparation, the HAM was mixed with VSMCs taken from rats for culture in vitro. Histological obserbation was done under inverted microscope and scanning electron microscope respectively. 20 rats were divided into two groups. Hemicystectomies were performed in 20 rats, and 10 of them were repaired with HAM grafts with VSMCs on the half bladder, the other 10 were repaired with HAM grafts without VSMCs as the control group. The rats underwent postoperative assessment of bladder volume at the 2nd,4th and 8th weeks, and the grafts were observed by light microscope at the 2nd,4th and 8th weeks after surgery. Results The physical and 1% trypsogen treated HAM was pure with hollows and undamaged collagen fibers. The VSMCs could grow, adhere to and differentiate on the surface of HAM and into the hollows. At the 2nd,4th and 8th weeks after surgery, the bladder volumes of the experimental group were not different significantly compared with those of the control group. Epithelialization and smooth muscle cells regeneration occurred with the infiltration of inflammatory cells in the 2nd week after grafting, and the HAM were absorbed. In the 4th and 8th weeks, it was difficult to delineate the junction between the host bladder and grafts by histology.Conclusion HAM can be used as the scaffold to construct tissue engineering bladder as it has good biocompatibility with VSMCs without disturbing the cell form and the graft can be absorbed quickly./P> Related Articles | Metrics

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DENSITY AND AREA OF LYMPH VESSELS IN HUMAN GASTRIC CANCER AND ITS RELATIONSHIP WITH LYMPH NODE METASTASIS 2008, 39 (4):  602-606.  doi: Abstract ( )   Objective To study the morphological characteristics of lymph vessels in human gastric cancer and to investigate whether lymph vessel density(LVD) and lymph vessel area are related with lymph node metastasis (LM). Methods Lymphatics in the normal, peripheral and central regions of 70 samples of primary human gastric cancer were investigated by immunohistochemical staining for D2-40. The relation between morphological characteristics of lymph vessel and LM and other established clinicopathological parameters were analyzed. Results Compared with the normal region of human gastric cancer, the peripheral region was found with a significantly highly LVD, and with significant decreases in the average area, average perimeter and total area of lymphatics. The LVD, average area and average perimeter of lymphatics in the peripheral region of human gastric cancer with LM significantly increased compared with those of human gastric cancer without LM.Conclusion Lymph angiogenesis exists in the peripheral region of human gastric cancer, and the lumina of newly formed lymphatics is small, therefore the lymphocinesia of that region is inadequate. Increased LVD and lymph vessel area were significantly related with LM, and may play an important role in detecting LM in human gastric cancer and the decision making for additional surgery. Related Articles | Metrics

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MORPHOLOGICAL OBSERVATION ON ELASTIC FIBERS IN THE REMODELING MYOCARDIUM OF RATS AFTER INFARCTION 2008, 39 (4):  607-610.  doi: Abstract ( )   Objective To investigate the changes and role of elastic fibers during the progression of ventricular remodeling after myocardial infarction (MI) in rats. Methods Male Sprague Dawley rats were induced by ligating the anterior descending coronary artery and were divided into the 2nd, 4th, 8th, 12th and 21st week groups. Elastic fibers were evaluated in the infarct region using Masson trichrome staining, Uana orcein staining and image analysis. The expressions of TNFα and IL-1β were also detected by immunohistochemistry. Results Elastic fibers could be found within the infarct region in the 2nd week after MI, increased with time and reached the peak during the 8th to 12th weeks, decreased or were absent in the 21st week. The expressions of TNF-α and IL-1β were also detected in the infarct zone and increased with time.Conclusion Elastic fibers were involved in infarct healing and may play an important role in pro Related Articles | Metrics

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OXYTOCIN INDUCES DIFFERENTIATION OF P19 CELLS INTO CARDIOMYOCYTES 2008, 39 (4):  611-613.  doi: Abstract ( )   Objective To investigate the effects of oxytocin (OT) on the differentiation of P19 cells into cardiomyocytes in vitro. Methods The P19 cells were cultivated in the medium containing different concentrations (1×10SUP>-7/SUP>,3×10SUP>-7/SUP> and 5×10SUP>-7/SUP>mol/L) of OT in suspension in dishes laid with a thin layer of agar for 4 days. Then the embryonic bodies (EBs) were transferred to coverslips in 24 well plates, allowed to adhere and be cultivated in the medium without OT for 10 to 12 days. The methods of immunocytochemical staining,transmission electron microscope(TEM) were used to identify cell differentiation. Results Some border cells in the peripheral outgrowth of the experiment groups became elongate and radial, and they were αsarcomeric actin (αSCA) and cardiac troponin T(cTnT) positively stained. The positive cells percentages the experiment group 1 (1×10SUP>-7/SUP>mol/L OT) were higher than those of other groups (P<0.01). The results of TEM indicated that the differentiated cells had the cha Related Articles | Metrics