Transplantation Of Genetically Modified Human Neural Stem Cells Contributes To Repair And Recovery From Spinal Injury In The Adult Rabbits

Up to now, we still lack effective medicine or means in clinic for the spinal cord injury( SCI) that lead to permanent paralysis of tens of thousands yang people every year worldwide. Resent development of neural stem cells (NSCs) raised hopes for the recovery and therapy of SCI. Much research showed, the keys of nerve losing the ability of renewing after SCI is lacking of potential cells and suitable tiny-environment. NSCs is one kind of neural precursor cells with dividing and differentiating potency. It may be clinically useful in generateing cell types that have been damaged in SCI. Animal studies demonstrated that NSCs provided such a sence of optimism ever before for transplantation treatment in SCI. It not only rises to the functions of bridging and nourishing, but also can complement and replace the damaged nerve cells, can still secrete factors to regulate and improve the tiny-environment. Combining together with the gene treatment, the NSCs can provide more ideal tiny-environment, stimulate nerve growth. At the first part of the experiments, we studied the isolation, cultivation and differentiation of NSCs from rats and humen embryo. At the second part of the experiments, we withdrew the human brain derived neurotrophic factor(BDNF), then, we constructed the recombinant retrovirus plasmid PLEGFP-BDNF using the hBDNF gene and PLEGFP vector. Because of some reasons in technique, we failed in producing out large quantity of BDNF- PLEGFP virus. Hence, we directly transfected the human neural stem cells(hNSCs) making use of the retrovirus taking the gene of EGFP. At the third part of the experiments, we made Cross-cutting injury models of rabbits at T9 vertebra level. The hNSCs was grafted to the injury site of spinal cord. The neural morphological repairing and functional recovery were investigated.Part â…  The Purification and Characterization of Embryonic Neural Stem Cells of Human and RatsObjective To isolate NSCs from cortex of rat and human embryos for setting up the long-term culture system of rat and human neural stem cells in vitro, so as to offer enough NSCs for transplantation therapy and geneticl modification. Methords NSCs were isolated from cortex of rat embryos(14 days) and human fetuses aged from 10 to 20 weeks following abortion. It was puried and characterized after being cultured some time. The limiting dilution with clonal expansion as neurospheres was operated, the growth curve of continually passaged NSCs was examined, the morphological changes, growth condition, differentiation status and immunohistochemistry features was observed. Results The rat and human neural stem cell line from fresh embryonic cortex at E10 to E20 weeks was established, the cells could be cloned and passed continuously, expressing Nestin antigen. After induced to differentiate, it expressed phenotypic markers for neurons and astrocytes. It had been passaged continuously 32 and 17 passages(10 months) respectively keeping strengthened capacity of proliferation in our labs. Conclusions NSCs could being isolated from rat and human embryonic tissues, it retained the biological features of stem cells after long-term culture in vitro. It could be conveniently applied to transplantation therapy and geneticl modification.Part II Construction of Recombinant Defective Retrovirus Vector With Human BDNF Gene and Genetical Modification of Human Neural Stem CellsObjective Constructing the recombinant retrovirus vector PLEGFP-BDNF to prepare further expression and transfection in cells for transplantation. To observe the boilogical changes after enhanced green fluorescent protein (EGFP) gene was transfected into the hNSCs by using retroviral in vitro.Methords The primers specific for the full-length BDNF coding sequence was designed and synthesized. The BDNF coding sequence was directly amplified from human genomic DNA by using PCR and inserted into vector PMD18-T. The recombinant DNA was transformed into the host cells E.coli DH5 a to obtain the positive clone PMD18T-BDNF. The restriction enzyme analysis and DNA sequence detection confirmed that the inserted fragment of clone is the full-length BDNF coding sequence. The hBDNF DNA fragment was recovered from the clone and ligated with eukaryonic expression vector PLEGFP plasmid to construct the recombinant expression plasmid PLEGFP-BDNF. The E.coli JM109 transformed with PLEGFP-BDNF was induced. The positive clone was screened on LB plates containing ampicillin andidentified by restrictive enzymes digestion, PCR amplification and DNA sequence detection. The retroviral-mediated EGFP gene was used to transfect hNSCs so as to observe the biological features and protein expression in vitro Results The recombinant expression plasmid PLEGFP-BDNF could express and amplify in E.coli JM109. The DNA sequence was in accord with standard hBDNF.These hNSCs can be genetically engineered to express foreign transgenes in vitro. It still remained undifferentiated and had self-renewing capacity and potential multipotency of differentiation after transfected. Conclusion PLEGFP-BDNF was constructed successfully. The hNSCs transfected with retroviral-mediated EGFP gene reserved the essential features of NSCs. It offered a effective instrument for further gene modification and transplantation therapy research.Part HI Transplantation of Genetically Modified Human Neural Stem Cells Contributes to Repair and Recovery From Spinal InjuryObjective To investigate the biological activity and effects on neural morphological repairing and functional recovery after hNSCs with retrovial-mediated EGFP gene transplanted to models of spinal cord injury in rabbits. Methords Cross-cutting injury to rabbits spinal cord was produced at T9 vertebra level, Neural morphological repairing and functional recovery were investigated after hNSCs grafted to the injury site of spinal cord in rabbits.Results These genetically engineered hNSCs were viable, migrated, and differentiated into neurons and glia after transplanted to the adult rabbits models of spinal cord injury, as well as improving motor neural regeneration, morphological repairing and functional recovery compared to the control, conclusion Transplation of human neural stem cells could improve neural morphological repairing and functional recovery following spinal cord injury. Neural stem cells'transplation is an effective approach for treatment of spinal cord injury.