An Experimental Study On Induced Differentiation Of Fetal Spinal-derived Neural Stem Cells In Vivo And Vitro And Optimal Time, Effect Of Them Transplanted For Preventing Denervated Muscles From Atrophy

It is a challenge for hand surgeons and neurosurgeons to delay intrinsic muscle atrophy of hand resulted from lower trunk of brachial plexus,median nerve and ulnar nerve injury.Function loss of hand intrinsic muscle means the core function loss of upper extremity,which brings a lot of burden of mind and economy for patients,their family,and society.So,strengthen of basis research and exploration of the nature of denervated muscle atrophy have considerable theoretical and practical importance.In recent years,those results were found neural stem cells transplanted into mammalian nervous system,especially the central nervous system,such as brain can survive, differentiate into neurons and glial cells and repair some functional deficits by replacement the damaged neurons and construction of new neural circuits.In this study,SD rats whose tibial nerves were transected were used as animal model,we explored the differentiation characteristic of neural stem cells in vitro and in vivo, whether they can differentiate into cholinergic neurons induced by neurotrophin-3(NT₃).We also evaluated the therapeutic effect of transplantation of NT₃-gene-modified neural stem cells on recovery of denervated skeletal muscle atrophy and motive function.At the time we confirmed the optimal time of the spinal cord-derived neural stem cells transplantation for preventing the skeletal muscles from denervated atrophy.The new strategies for preventing the skeletal muscles from denervated atrophy and recovering their motive function would be provided to hand surgeons and neurosurgeons by the results of this study.Objective:(1) To explore the isolation,cultivation,identification,proliferation characteristic and differentiation in vitro.(2) To assess whether the embryonic spinal-derived neural stem cells can differentiate into cholinergic neurons induced by NT₃.(3) To evaluate whether the NT₃-gene-modified neural stem cells have much better therapeutic effect for the prevention of denervated skeletal muscle atrophy and recovery of motive function.(4) To determine the optimal time of the spinal cord-derived neural stem cells transplantation for preventing the skeletal muscles from denervated atrophy. Methods:(1) The neural stem cells were separated from spinal cord of P14-16 day SD rats' embryo mechanically and were cultivated in a serum-free tissue culture medium DMEM/F₁₂ containing N₂ supplement,B₂₇,basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF) for their primary neural stem cells and passages.Single cell cloning was undertaken in order to purify the cells,the technique of immunoflurenscence cytochemistry was applied to identify the neural spheres with the marker of nestin.At the same time the technique was also applied to identify the differentiated cells with the marker ofβ_â…¢-tubulin for neurons and glial fiber acidic protein(GFAP) for astrocytes.These cells were observed by their morphological changes in the process of proliferation,differentiation of the neural stem cells.(2) The neural stem cells were obtained from the spinal cord of P14-16 day SD rats' embryo.The differentiation of neural stem cells was induced by NT₃ in vitro: In experimental group,the spinal cord-derived neural stem cells were cultivated in the medium(DMEM/F₁₂ containing fetal bovine serum(FBS)) for the differentiation of neural stem cells adding 20ng/ml NT₃;in control group,the spinal cord-derived neural stem cells were cultivated in the same medium not adding NT₃.The technique of immunoflurenscence cytochemistry was applied to identify the differentiated cells with the marker ofβ_â…¢-tubulin for neurons and choline acetyltransferase(ChAT) for cholinergic neurons 48 hours,1 week and 3 weeks following the induction.The differentiation of neural stem cells was induced by NT₃ in vivo:In order to have animal model which was denervated gastrocnemius of adult SD rats,the tibial nerves of them were transected.40 adult SD rats whose right tibial nerves were transected,were divided into 2 groups randomly:neural stem cells transplanted group(control group) and neural stem cells induced by NT₃ transplanted group(experimental group).There were 20 rats in each group and they were divided into 2 subgroups randomly:4 weeks subgroup and 8 weeks subgroup following neural stem cells transplantation.In experimental group the suspension for the differentiation of neural stem cells adding 20ng/ml NT₃ of neural stem cells(5×107/ul,0.02ml) were injected into the distal stump of tibial nerves with microsyringe made by ourselves(outer diameter 200μm),in control group the same dose of the suspension not adding NT₃ were injected into the distal stump of tibial nerves by the same way.The rats were anesthetized again and the transplanted part of tibial nerves were harvested 4,8 weeks following the neural stem cells transplanted.The technique of immunoflurenscence cytochemistry was applied to identify the differentiated cells with the marker ofβ_â…¢-tubulin for neurons and choline acetyltransferase(ChAT) for cholinergic neurons with frozen sections of tibial nerves.(3) The neural stem cells were obtained from the spinal cord of P14-16 day SD rats' embryo.The animal model which was denervated gastrocnemius of adult SD rats was obtained from the tibial nerves of them were transected.The lentivirus expression vector of NT₃ gene was constructed then the spinal-derived neural stem cells were transfected with the NT₃ gene.Whether the neural stem cells were transfected by the NT₃ gene was detected by RT-PCR and expression of NT₃ gene was detected by Western blot.Differentiation of the neural stem cells transfected by the NT₃ gene was observed 3,5 days following tansfection.90 adult SD rats whose right tibial nerves were transected,were divided into 3 groups randomly:neural stem cells transplanted group,neural stem cells induced by NT₃ transplanted group and NT₃-gene-modified neural stem cells transplanted group.There were 30 rats in each group and they were divided into 3 subgroups randomly:1 week subgroup,4 weeks subgroup and 12 weeks subgroup following neural stem cells transplantation.The suspension of the neural stem cells(5×107/ul,0.02ml) was injected into the distal stump of tibial nerves with microsyringe made by ourselves(outer diameter 200μm) by the previous described method.The rats were anesthetized again and the transplanted part of tibial nerves were harvested 1 week following the neural stem cells transplanted.The technique of immunoflurenscence cytochemistry was applied to identify the survival of the neural stem cells with the marker of nestin and GFP.The rats were anesthetized again and the transplanted parts of tibial nerves were harvested 4 weeks following the neural stem cells transplanted.The technique of immunoflurenscence cytochemistry, histochemistry and image analysis was applied to assay the differentiation of the neural stem cells.The rats were anesthetized again 12 weeks following the neural stem cells transplanted.At first the rats were taken the EMG examination,then the transplanted part of tibial nerves and the both gastrocnemius were harvested.These specimens were deal with the same method described previously.(4) The neural stem cells were obtained from the spinal cord of P14-16 day SD rats' embryo.The animal model which was denervated gastrocnemius of adult SD rats was obtained from the tibial nerves of them were transected.90 adult SD rats whose right tibial nerves were transected,were divided into 9 groups randomly and there were 10 rats in each group. The suspension of the neural stem cells(5×107/ul,0.02ml) was injected into the distal stump of tibial nerves with microsyringe made by ourselves(outer diameter 200μm) by the previous described method 0,1,2,3,4,6,8,12,16week(s) following transection of tibial nerves.Rats were euthanized 4 weeks and the transplanted part of tibial nerves and the both gastrocnemius were harvested 4 weeks following the neural stem ceils transplanted.The technique of immunoflurenscence cytochemistry and image analysis was applied to count the quantity of neurons with frozen sections of tibial nerve,at the same time the technique of histochemistry and image analysis was applied to measure gastrocnemius muscle fiber cross-sectional area.Results:(1) A number of neural stem cells were obtained from the spinal cord of P14-16 day SD rats' embryo by means of the mechanical dissociation and the culture with serum-free medium.The obvious neural sphere can be observed in about 5-7 days and can be maintained proliferation in vitro for more than 8 weeks.The neural sphere can be obtained again by cultivation of single cell cloning.The cells were Nestin positive showed there were neural stem cells,and the differentiated cells wereβ_â…¢-tubulin and GFAP positive showed there were neurons and glial cells.These confirmed the cells obtained from the spinal cord of P14-16 day SD rats' embryo were neural stem cells.(2) In experimental group ChAT positive cells were detected 48 hours,1 week and 3 weeks following induction of NT₃ in vitro;no ChAT positive cells were detected in control group butβ_â…¢-tubulin positive cells were detected.In experimental group ChAT positive cells were detected 4 weeks and 8 weeks following induction of NT₃ in vivo;no ChAT positive cells were detected in control group butβ_â…¢-tubulin positive cells were detected.(3) The size of RT-PCR product of the positive cell clone was 750bp and the positive signals of protein were obtained by Western blot.GFP and nestin positive cells were detected in all groups 1 week following neural stem cells transplanted;GFP and ChAT positive cells were detected in NT3-gene-modified group and NT3+NSC group 4 weeks following neural stem cells transplanted and their quantity is different(p＜0.01).But GFP and ChAT positive cells were detected only in NT3-gene-modified group 12 weeks following neural stem cells transplanted.Improvement in histology and electrophysiology in NT₃-gene-modified neural stem cells transplanted group was much better than in neural stem ceils transplanted group and neural stem cells induced by NT₃ transplanted group(p＜0.05).(4) Quantity of neurons and CSA of gastrocnemius in the group which neural stem cells were transplanted 1 week following tibial nerves transected were much better than other groups(p＜0.01),the second is the group which neural stem cells were transplanted 6 weeks following tibial nerves transected(p＜0.05).Conclusion:(1) A number of neural stem cells were obtained from the spinal cord of P14-16 day SD rats' embryo by means of the mechanical dissociation and the culture with serum-free medium and they can differentiate into various types of neural cells such as neurons and glial cells.(2) The neural stem cells can differentiate into cholinergic neurons induced by NT₃ in vitro and in vivo.(3) The neural stem cells can be transfected by NT₃ gene with lentivirus as vector.NT₃-gene-modified neural stem cells can survive and differentiate into neurons in vitro and in vivo and they have much better therapeutic effect for the prevention of denervated skeletal muscle atrophy and recovery of motive function.(4) The optimal time that spinal cord-derived neural stem cell transplantation for preventing skeletal muscle denervation atrophy is 1 week following nerves transection and the second is 6 weeks following nerves transection.