Objective To investigate whether the skin-derived neural stem cells inducted by the culture could survive,differentiate and migrate in the lesioned site of rat spinal cord.Methods The skin of new born rat trans-gene of green fluorescence protein(GFP) was applied to be dissociated into cells,cultured and inducted to proliferate in vitro.Skin-derived neural stem cells were identified by immunocytochemistry staining.Then,skin-derived neural stem cells inducted by the culture were transplanted into the lesioned site of rat spinal cord hemisection.Thirty days and sixty days after the operation,the survival,migration and differentiation of transplanted cells were observed by immunocytochemistry staining. Results Ten days at cultured skin-dissociated cells,many cell-spheres had been formed by the proliferation of suspensive growing cells.These cell-spheres showed nestin positive staining of immunocytochemistry.It suggestes that the cell-spheres are neurospheres.In vivo,many transplanted skin-derived neural stem cells with GFP were observed in the lesioned area of spinal cord.Some transplanted cells migrated into host spinal cord tissue far away from the lesioned area.Some transplanted surviving cells showed nestin,MAP2 and GFAP positive staining of immunocytochemistry separately.Conclusion Skin-derived neural stem cells inducted by the culture may survive,migrate and differentiate into neuron-like cells and astrocyte-like cells in injured spinal cord of rat.

Objective To explore the effects of DU meridian electroacupuncture on the survival,differentiation and immigration of neural stem cells(NSCs) transplanted in the injured site of rat spinal cord transection. Methods Twenty adult SD rats were divided into NSCs transplanted 14d group(NSCsl4d group),DU meridian electroacupuncture plus NSCs transplanted 14d group(EA+NSCs14d group),NSCs transplanted 30d group(NSCs30d group) and DU meridian electroacupuncture plus NSCs transplanted 30d group(EA+NSCs30d group).T10 spinal cord segments of all animals were completely transected.The rats of EA+NSCs14d group and EA+NSCs30d group were treated with DU electroacupuncture at 5 days after operation.The sacrifice times of the rats were respectively at 14 days and 30 days after operation.The spinal cords were taken for observing the survival,differentiation and immigration of neural stem cells transplanted in the injured site of rat spinal cord transection. Results 1.The surviving number of transplanted NSCs in EA+NSCs14d group or EA+NSCs30d group was more than that of NSCsl4d group or NSCs30d group,and the surviving number of transplanted NSCs in EA+NSCs30d group and NSCs30d group was less than that in EA+NSCs14d group and NSCs14d group.2.Some transplanted NSCs at injured site of transected completely spinal cord and neighboring tissue showed microtubule association protein 2(MAP2) positive staining in EA+NSCs30d group or NSCs30d group.3.Many transplanted NSCs at injured site of transected completely spinal cord and neighboring tissue could be observed to show glial fibrillary acidic protein(GFAP) positive staining in EA+NSCs 30d group or NSCs 30d group.4.The immigrating distance of transplanted NSCs toward caudal tissue of injured site of spinal cord was longer in EA+NSCs 14d group and EA+NSCs 30d group than that in NSCs 14d group and NSCs 30d group.Conclusion DU meridian electroacupuncture may promote the survival of neural stem cells transplanted in injured site of rat spinal cord transected completely,and these cells can differentiate into MAP2 or GFAP possive cells.DU meridian electoacupuncture may affect the immigrating direction of neural stem cells transplanted at injured site of spinal cord toward host spinal cord tissue.

Objective To investigate the intervening effects of Ganoderma spore on the decrease of cell proliferation and neuronal survival in the hippocampus of fetal and postnatal rats induced with gestational hypertension. Methods Fourty SD pregnant rats were divided into four groups including the control group,Nw-nitro-L-arginine methylester(L-NAME)+distilled water(DW) group,L-NAME+L-Arginine group and L-NAME+Ganoderma spore(GS) group.The hippocampal tissue of the brain was detected by immunohistochemistry,Western blotting,RT-PCR,flow cytometry and electron microscopy. Results After the application of L-NAME,the expressions of hypoxia inducing factor-1?(HIF-1?) and vascular endothelial growth factor(VEGF) were increased at the hippocampus of E21 brain and continued up to P30 brain.The microvessel density of E21 hippocampus was increased and the structural abnormalities of blood capillary at P30 hippocampus were showed.The cell proliferation was decreased at E21 hippocampus and so was the neuronal number at P30 hippocampus.With the administration of Ganoderma spore,HIF-1? and VEGF were down-regulated at E21 hippocampus and were not detected at P30 hippocampus.The microvessel density of E21 hippocampus reached a normal level and the blood capillary ultrastructure of P30 hippocampus was restored.The cell proliferation of E21 hippocampus and neuronal number of P30 hippocampus were recuperatively increased.Conclusion Ganoderma spore may prevent the decrease of cell proliferation and neuronal survival in the hippocampus of fetal and postnatal rats induced with gestational hypertension.

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.

Nerve growth factor (NGF) can promote the outgrowth of neurites of the target ganglia. In order to further explore the relationship between this effect and the synthesis of RNA and DNA in the neurons, an autoradiography of 3~H-uridine and 3~H-thymidine was used. Superior cervical ganglia (SCG) from newborn rats were cultivated by Maximow's double coverslip method. All cultures were divided to one group of cultures a crude preparation of NGF was added to the medium and another group without NGF served as control. Before tissue culture was stopped, the. covership cultures were transferred to thelabeling-medium and incubated, and then they were fixed, and cut into serial sections and subjected to autoradiographie processes. The results show that the percentage and the level of grains of neurons labeled by 3~H-uridine in the NGF group are higher than that of control. Moreover, before the growth rate of neurites reaches a peak, the level of grains of neurons labeled by 3~H-uridine in the NGF group is obviously increased. The evidence suggests that NGF can promote the synthesis of RNA in neurons of SCG, which has a direct bearing on the quick outgrowth of neurites. In the experiments with 3~H-thymidine incorporation, that the NGF may promote the synthesis of DNA in some neurons of the third day SCG in vitro was also observed.

Objective To explore a fast,simplified and economical method for Schwann cells(SCs) cultured and purified in vitro.In this way,a stable and reliable cell sources can be provided in order to study SCs transplanted in vivo. Methods SCs cultures were prepared from the sciatic and brachial nerves of 3 to 5-day-old SD neonatal rats with double enzyme digestion method to acquire dissociated cells and one enzyme digestion method to acquire incomplete digested tissues. Results With the same number of neonatal rats,one enzyme digestion(hemi-explant)method acquired at least two times more SCs than double enzyme digestion(single cell) method did and saved at least 40 minutes.96% of S-100 positive SCs cultured were shown by immunohistochemical staining.Conclusion When the one enzyme digestion method(hemi-explant) is used to culture SCs,sufficient SCs with qualified purity can be acquired in a short time.

Objective To detect the expression and the biological activity of a recombinant adenovirus expression vector carrying human neurotrophin-3 (NT-3) receptor TrkC gene (Adeno-TrkC) in neural stem cells. Methods The expression of TrkC mRNA in 293 cells infected with Adeno-TrkC was detected by RT-PCR, and the expression of TrkC protein in neural stem cells infected by Adeno-TrkC was detected with immunocytochemistry and Western blotting. The effects of human neurotrophin_3 (NT-3) on the neural stem cells infected by Adeno-TrkC differentiating into neuron-like cells and astrocyte-like cells in vitro were observed. Results The transcription of TrkC mRNA and the expression of TrkC protein was detected in 293 cells and neural stem cells infected by Adeno-TrkC. This kind of TrkC was able to make more neural stem cells differenting into neuron-like cells in vitro with its ligand NT-3 and the percentage of neuron-like cell’s differentiation was 55^2% which was higher than that of other control groups.Conclusion TrkC protein is expressed in neural stem cells transfected with Adeno-TrkC and is able to make neural stem cells differenting into neuron-like cells. The present study may provide the basis on gene therapy of central nervous injury using further NT-3 and TrkC.

10kD fraction from operated side of two groups of animal was tending towards increasing, especially the approximate 67kD protein content from operated side of morphine group of animal.\;

Objective To explore the effects of morphine on the terminal of primary afferent fiber distributing in spinal lamina Ⅱ after the sciatic nerve crush. Methods The positive reactive areas of fluoride-resistant acid phosphatase(FRAP) at spinal lamina Ⅱ were measured by the FRAP histochemistry and microcomputer image analysis techniques, after sciatic nerve was injured 15 days and 30 days both in morphine and control groups of rats. Results The positive reactive areas of FRAP at spinal lamina Ⅱ were depleted on different degree in two groups of rat after sciatic nerve was injured. The positive reactive areas of FRAP were greater 40% on injured sciatic nerve in 30 days than in 15 days in control group. In morphine group, the positive reactive areas of FRAP were larger 22% on injured sciatic nerve in 30 days than in 15 days; simulaneously also were bigger 19% than on injured sciatic nerve 30 days of control group.Conclusion The positive reactive areas of FRAP at spinal lamina Ⅱ show recovering enlargement as the surviving time lengthens in both groups of rats injured sciatic nerve.Morphine may enhance the positive reactive area of FRAP at spinal lamina Ⅱ in rat of sciatic nerve crush.