AIM　To Detect the varying of gene expression in cultured dorsal root ganglion cells treated by Nerve Regeneration Factor and explore its molecular mechanism promoting to nerve growth. METHODS　By RT-PCR, the change of gene expression in GAP-43 and NF-L on the cultured DRG cells with Nerve Regeneration Factor were studied during 4 h, 12 h, 24 h. RESULTS　The expression of GAP-43 and NF-L were increased by Nerve Regeneration Factor on DRG′s culture. CONCLUSION　This study indicated Nerve Regeneration Factor may up-regulate the gene expression associated nerve growth on cultured nervous cells.

Objective To observe expression of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) in nerve and dominated muscle of rat after injury of sciatic nerve.Methods 12 female SD rats were divided into 4 groups, right sciatic nerve was squeezed by using forceps for 0.5hr, 1hr, 2hr and 5hr respectively. Then right sciatic nerve and gastrocnemius was isolated and RNA was extracted by using Trizol reagent and meanwhile, the left sciatic nerve and gastrocnemius was used as a normal control. NOS expression was detected by using RT PCR and RNAase protection assay (RPA), and GAPDH was used as an internal standard. The density of PCR and RPA bands was determined by using NIH image software.Results 2 NOSs did not vary in nerve tissue in 4 groups, but in muscle, nNOS increased in 1h group, decreased in 2h group and increased in 5h group; iNOS decreased in 1h and 2h groups but increased in 5h group when compared to normal control.Conclusion Injuring of nerve does not effect NOS expression itself within short period, but effects the dominated muscle via the transmission of non NOS nerve signal.

Objective To establish a method of neurons and glial cell culture from embryonic Gekko japonicus cerebral cortex. Methods Embryonic(E15) pallium was dissociated and digesting by trypsin.After counting,cells were seeded in culture flask.The glial cells were obtained by using differential adhesion potential combined with successive passage purified methods,and neurons were obtained by using neurobasal medium supplemented with B27.The cells were fixed and analyzed with immunohistochemic assay. Results After tetra-generation,GFAP positive cells were more than 95% in glial cells cultured condition;Neurons grew well in neurobasal medium,and NF and MAP-2 positive signals co-localized on neurons.After cultured for 10 days,the percentage of neurons was more than 95%.Conclusion The methods of isolation,culture and purification for embryonic Gekko japonicus cortical neurons and glial cells were established and it might be a valuable cell model to further investigate the central nerve system in Gekko japonicus.

The special trichrome stain and immunocytochemical stain were used to show neurites, Schwann's cells in cultured pe-ripheral nerve tissue. The dorsal root ganglia(DRG) of rat were cultured on polypyrrole membrane for 2 weeks. Then, the cul-tured speciments were stained by special stain, which was composed of hematoxylin, fast green FCF. ehromotrope 2R and phos-photungstic acid; or by immunocytochemical stain with anti-S-100 protein and anti-neurofilament antibodies. In the specialtrichrome stained specimen the long processes from DRG were stained aquamarine blue; part of the cell nuclei on the processes orpolypyrrole membrane were stained red or purplish red, and the cytoplasm ashen. We testified that the long processes from DRGwere neurites and the cells which were purplish red nuclei and ashen cytoplasm were Schwann's cells in immunocytochemicalstain. The special staining could differentiate neurites and Schwann's cells in cultured peripheral nerve tissue.

Objective To observe the protective effects of salidroside on glutamate-induced injury in cultured hippocampal neurons.Methods Primarily cultured hippocampal neurons from fetal Wistar rat were incubated with salidroside(10,20 and 40mg/L) for 24 hours,then glutamate(125?mol/L) was added for 15 minutes to induce injury.Cell viability was detected by MTT assay,the vigor of LDH was determined by biochemistry method,the apoptosis rates were anallyzed using Annexin V-FITC and PI labelling and Hoechst 33342 staining and flow cylometric assay.Fluorescent intensity of intracellular free calcium was observed with laser scanning confocal microscopy(LSCM).Results After the pretreatment with salidroside for 24 hours,the increases of LDH vigor and apoptosis rates and the decrease of cell viability caused by glutamate were resisted obviously.Salidroside inhibited the increase of Ca~2+ in cytoplasm significantly.Conclusion Salidroside can significantly resist the injury induced by glutamate.The neuroprotective activities of salidroside can be related to its ability to reduce Ca~2+ overload in cytoplasm.

Objective To investigate the biocompatibility of bone marrow stromal cells(BMSCs) of mice in vitro with silk fibroin materials and to explore a novel scaffold material to fabricate tissue-engineered nerve with introduction of BMSCs.Methods BMSCs were typically isolated from other cells by adherence to plastic.The mice-derived bone marrow stromal cells were cultured on the substrate of silk fibroin fibers and the cell attachment and growth during culture was observed by using light and electron microscopy.Mice-derived BMSCs were also cultured in the silk fibroin extract fluid.The cell ultrastructure was observed by transmission electron microscopy.MTT test was used to detect cell viability in different media for 12,24,48,72 hours and 7 days respectively(the test was repeated 12 times for each group).Flow cytometry was employed to detect BMSCs cell cycle and phenotypes(the test was repeated 3 times).Results BMSCs cells were tightly attached to the silk fibroin fibers and grew along the silk fibroin fibers;some of them enwrapped the silk fibroin fibers and they exhibited either a spherical or spindle shape.The results of transmission electron microscopy,MTT test and flow cytometry analysis showed that there was no significant difference between BMSCs cultured in the silk fibroin extract fluid and those in plain IMDM medium in their morphology,cell viability,proliferation and phenotypes.Conclusion These data indicate that silk fibroin has good biocompatibility with BMSCs and is also beneficial to the survival of BMSCs without exerting any significant cytotoxic effects on their phenotype;thus it's a potential scaffold material to fabricate tissue-engineered nerve with introduction of BMSCs.

Objective To evaluate the expressive variety of NGF gene in injured sciatic nerves of rat. Methods For hemi-quanti- tative analysis, RT-PCR method was used in the detection of the levels of NGF mRNA in the distal stumps at 1 day and 1, 2, 4 weeks following transection of sciatic nerve. Results The 1evel of NGF mRNA was low in the normal sciatic nerve. But it was increased in the distal stump after sciatic nerve transection. The biphasic increase in NGF mRNA was characterized by a first very rapid and transient increase peaking at ist day after lesion, than a second prolonged and slow elevation starting around ist weed after lesion. Conclusion The process in NGF gene expression after sciatic nerve lesion was biphasicly increased.

Objective\ Clone and sequence choriocarcinoma related gene T26. Method\ Choriocarcinoma related gene T26 was ligated into PGEM\|T vector using T\|A clone method.PGEM\|T\|T26 was digested with EcoRⅠ and sequenced,comparing the T26 sequence with GenBank. Results\ The sequence of choriocarcinoma related gene band T26 showed more than 99% identity with the 3' end of human scar,rps4 and ccg2 genes. Conclusion\ The gene T26 besides scar,rps4 and ccg2 genes were related with the oncogenesis of choriocarcinoma. [

Objective To observe CNTF gene expression and expressive variety during postnatal development in rat spinal cord. Methods The spatial expression of CNTF mRNA in spinal cord was examined by in situ hybridization with dig\|CNTF cDNA probe.Reverse transcription\|polymerse chain reaction(RT\|PCR),as hemi\|quantitative method was used to investigate the expressive variety of CNTF mRNA levels in spinal cord during postnatal development. Results Hybridized signals of CNTF mRNA was found only in part of glial cells on the peripheral region of the ventral and lateral white funiculi of spinal cord,but could not be detected in the gray matter of spinal cord.The expression of CNTF mRNA in spinal cord appeared with a lower level on the first postnatal day.It increased significantly at postnatal 15th days,the expression of CNTF mRNA reached the peak at 30th days,and it began to decrease on the 60th days. Conclusion This study indicated that CNTF mRNA might be expressed in part of glial cells in the white matter.There was expression in spinal cord on the 1st day after birth and the expression levels of CNTF mRNA possessed the changeable character during spinal cord development.

Objective To dynamically observe photodamage of subcellular sites by use of laser scanning confocal microscopy (LSCM). Methods The samples were divided into four groups. Murine lung endothelial cells were subcultured and incubated with HMME for 24 hours. Then the cells were stained with rhodamine-123 for demonstration of mitochondria. LSCM was applied and organelle-cell fluorescence intensity ratio analysis was adopted to study the intracellular distribution of HMME. Then dynamic fluorescence images sequence of rhodamine-123 was collected. Results Rhodamine-123′s fluorescence images of cell sample with HMME was changed gradually during irradiation: the typical characteristic of mitochondria disappeared gradually with decreasing fluorescence intensity. The fluorescence of rhodamine-123 was diffused and distributed in nuclear, while rhodamine-123′s fluorescence images of cell sample without HMME was not changed. Conclusion Mitochondria and nucleus are photodamage sites by HMME-PDT; LSCM can be applied in dynamic observation of photodamage of subcelluar sites.