An Experimental Study On Differentiation Of Neural Stem Cells In Vivo And Vitro And Transplantation Of Neural Stem Cells Delaying Denervated Muscle Atrophy

PartⅠAn experimental study of proliferation and differentiation of neural stem cells from GFP transgenic rats in vitroObjective To investigate the biologic activity of cultivation and differentiation of neural stem cells from the spinal cord of embryonic GFP transgenic rats and a method to induce neural stem cells to differentiate neurons in vitro.Methods 1．Neural stem cells were separated from P14～16 day spinal cord of GFP transgenic rats mechanically and were cultured and induced to differentiate in vitro. The indirect immunofluorescente cytochemistry were used to identify the neural stem cells and the differentiated cells with the marker of Nestin for neural stem cells,β-tubulinⅢfor neurons, GFAP for astrocytes and NG2 for oligodendrocytes. 2．After neural stem cells were passed over three generations, Laminin and Heparin were added in the original medium . The stem cells were continued to suspended and cultured for 5 days in vitro and then they were cultured to attach and differentiate in NSC-differential medium. After two weeks, immunofluorescente cytochemistry were used to identify neurons and the ratio of neurons was counted. The synaptophysin expressed from the differentiated cells was half-quantitated by use of reverse-transcriptase-polymerase chain reaction (RT-PCR). The control group included NSCs culturing with normal methods in vitro and differentiated cells.Results 1．The GFP-NSCs which could proliferate stabilized in vitro were obtained from the spinal cord of embryonic GFP transgenic rats by means of the mechanical dissociation and they could produce three kinds of neural cells, including neurons, astrocytes and oligodendrocytes, when being induced to differentiate in vitro.2．The improved method of culturing and inducing NSCs increased the ratio of neurons differentiating from NSCs (from below 7% to above 20%).Conclusions 1．The biological characteristics of NSCs derived from the spinal cord of embryonic GFP transgenic rats is satisfactory and similar as normal NSCs. 2．The improved method inducing NSCs in this research may significantly increase the ratio of neurons differentiating from NSCs and may be valuable for further research about NSCs differentiation and NSCs transplantation for retarding the atrophy of denervated skeletal muscle in vivo.PartⅡAn experimental study of survival and differentiation of neural stem cells from GFP transgenic rats after transplantation in peripheral nerveObjective To explore the survival and differentiation of NSCs from GFP transgenic rats manipulated by different ways after transplantation in peripheral nerve.Methods 1．The Lentivirus vector including NT3 gene was constructed and transfected into GFP-NSCs. 2．Establishing an animal model of F344 rat with apocoptic tibial nerve. Three groups of neural stem cell with different interventions were made into Single-cell suspensions and transplanted to the distal part of the tibial nerve (Group1: neural stem cell. Group2: neural stem cell which was induced to motor neuron in vitro. Group3: neural stem cell which was induced to motor neuron in vitro and transfected neurotrophic factor NT3．). At 4 and 12 weeks after implantation, the distal part of tibial nerve was harvested and identified for neurons, motor neurons, neuroglia cells, axons and synaptophysins with specific markers by means of indirect immunofluorescent staining respectively. Similarly, muscles were obtained and identified acetylcholine receptors and synaptophysins with specific markers by means of indirect immunofluorescent staining respectively.Results After GFP-NSCs were transplanted in tibial nerve, the stem cells survived and differentiated to astrocytes , oligodendrocytes ,neurons and motor neurons. And the differentiated cells sent out axons towards the denervated muscle. At 12 weeks after implantation, many synaptophysins gathered on the surface of denervated skeletal muscles and the form of postsynaptic membrane keep well and was similar as the one of normal membrane. After induced in vitro ,the proportion of neurons differentiated from GFP-NSCs was obviously increased and the differential time was advanced. The ratio of neurons differentiated from GFP-NSCs transfected NT3 gene was raised further. Among the three groups ,the ratio of neurons , the occurring time of motor neurons and the expressing intensity of synaptophysins in group 3 were best and those in group 2 were better.Conclusions 1．Neural stem cells from GFP transgenic rats can differentiate motor neurons in vivo after transplantation.2．The differential effect in vivo improved significantly after induced in vitro or transfected NT3 gene simultaneously. The results may be valuable for further research about NSCs transplantation for retarding the atrophy of denervated skeletal muscle in vivo.PartⅢAn experimental study of neural stem cell transplantation delaying denervated muscle atrophyObjective Discussing the effect of delaying denervated muscle atrophy after neural stem cell transplantation in the peripheral nerve.Methods Establishing an animal model of F344 rat with apocoptic tibial nerve. Three groups of neural stem cell with different interventions were made into Single-cell suspensions and transplanted to the distal part of the tibial nerve (Groupl: neural stem cell. Group2: neural stem cell which was induced to motor neuron in vitro. Group3: neural stem cell which was induced to motor neuron in vitro and transfected neurotrophic factor NT3．). The material was taken 4 weeks and 12 weeks after cell transplantation. Wet weight of triceps surae was measured. The denervated muscle was given HE staining and Mallory staining, while the frozen muscle biopsy tissue was given Immunofluorescence with postsynaptic membrane AchR specific antibody. The section area of gastroenemius fiber and the area of postsynaptic membrane were determined with image analysis software and taken statistic analysis.Results The decrease of wet weight, fiber section area and area of postsynaptic membrane appears in all groups with different degrees. However, the results of all three experiment groups were better than which of the control group. Among the experiment groups, group3 was better than group2 and group2 was better than groupl, which had significant difference after statistic analysis.Conclusions 1．The transplantation of embryonic spinal stem cells of GFP transgenic rat has taken the effect of delaying denervated muscle atrophy and taken an important position in keeping the normal appearance of postsynaptic membrane. 2．The effect of delaying denervated muscle atrophy will increase significantly after the neural stem cell induced to motor neuron in vitro or in the mean time transfected neurotrophic factor NT3．...