The Research Of Htert Gene And Rat Fetal Neural Stem Cells Immortalization

Some central nervous system disorders (such as Parkinson's disease, Huntington's disease, demyelinating disease and brain, spinal cord injury, etc.) is because of a group of damaged nerve cells, which seriously harm to human health and impact on people's quality of life. Traditional medication only can alleviate these diseases but not cure, and nerve system is sensitive to medicine and vulnerable to side-effects.In recent years, with the development of cell transplant technology, Neural stem cell transplantation has become effectively approach to treatment of these diseases.Neural stem cells are specifically primitive neuron cells in nervous system, and widely exist in many parts of mammalian central nervous system. They can generate all types of nerve cells through division, multiplication and differentiation, and self-maintenance and renew over lifespan. NSCs has become an ideal gene or medication carrier and cell-transplant donor for their self-renewal, multi-potential, lower immunogenicity, better migration and tissue integration properties, and so on. However, NSCs can appear replicative senescence when cultured in vitro f long period of time, and slowly proliferating and long passage period made them hardly to meet the needs of research and clinical application, which also hindered the process of transplantation. And immortalized NSCs/progenitor cells have show many advantages in transgenic therapy and research of transplantation in central nervous system, such as self-renew and quantity of proliferate, and stably expressing report gene for tracer labeling or therapy gene by genetic manipulation, and NSCs, immortalized by exogenous gene, still have potential to differentiate just as normal neural stem cells in vivo.In this study, a recombinant plasmid of pEGFP-N1-hTERT has been constructed and transferred into rat embryonic neural stem cells. And the following conclusions mainly drawn by fluorescence microscopy, CD133-positive cells of flow cytometry analysis, characteristic identification, cryopreservation and other aspects of a more systematic study:1. Established of rat embryonic of neural stem cell culture system Base on the analysis of NSCs cultured in different serum free mediumⅠandⅡ, the effect on neurosphers forming with different enzyme and the diversity of proliferation between floating culture and adherent culture, conclusion as follows:(1)We found that Serum-free medium SFCM I is a successful medium for culture rat fetal NSCs in vitro, and accelerate the NSCs proliferation and neurosphers formation in vitro, which meet the NSCs proliferate needs.(2)Our result shows that AccutaseTM is a perfect cell detachment solution when the NSCs neurospheres passing, The NSCs survival rates is around 92.6 per cent after digested. And you'd better beating upon gently and keeping neurospheres in a state of suspended digest, thus it will not only not only reduce the enzyme effect on NSCs and prevent the cells sticking together, which produced the higher survival rates and quantity, and also increased the number of neuorshpers formation.(3)It was found that the method of 1.5 per cent agarose anti-adherent is more appropriate for NSCs proliferating in vitro, compared with the method of coated plasticwarethe by gelatin, rat tail collagen and the poly-L-lysine.I culture method is more appropriate for neural stem cells in vitro.2. The rat fetal neural stem cell cryopreservation and differentiation in vitro Based on the analysis of cell survival rate and colony formation rate after cryopreservation in different protection medium, and NSCs differentiation with three cytokines NGF, BDNF and GDNF:(1)It is can be choose D-MEM/F12+10%DMSO medium supplied with 20 per cent of FBS or NBS when NSCs cryopreserved, and cryopreservation has no affect on the NSCs pluripotent.(2)The results show that when neural stem cells cultured in vitro supplied with 100 ng/mL of NGF, BDNF or GDNF lonely in the culture medium, they were induced to differentiation. When supplied with NGF or BDNF in culture medium, the NSCs were mostly induced to neurons. And the NSCs can differentiate to oligodendrocyte when supplied with GDNF.3. The construction and identification of pEGFP-N1-hTERT eukaryotic expression vectorHuman telomerase reverse transcriptase gene fragment of accuracy was confirmed by double enzyme digestion and DNA sequence analysis, and the positioning of hTERT gene has been observed in cell by GFP expression, and the construction of eukaryotic expression vector pEGFP-N1-hTERT has been verified. Recombinant plasmid can express fusion protein of hTERT and GFP in transferred NSCs. And the transfection efficiency and results could be observed timely by the expression of GFP. The construction of human telomerase reverse transcriptase eukaryotic expression vector is the foundation for the establishment of the further success of immortalized neural stem cells.4. The immortalization and identification of rat fetal neural stem cell linesBased on the observation of GFP expression in NSCs transferred recombinant plasmids, and the identification of multi-potential, analysis of RT-PCR and Western-blot, the proliferation and cell cycle distribution of CD133+ cells in neurospheres by flow cytometry analysis, we concluded that:(1)The rat fetal NSCs was passage 7 when cultured in vitro, and 4.0×107 cells were stored in liquid nitrogen. And the NSCs, transferred recombinant plasmid, was up to passage 12, and 3.35×107 cells were stored.(2)In this test, rat fetal NSCs transferred with the recombinant plasmid pEGFP-N1- hTERT still have the growth characteristics and multi-potential, that is, they can be formed neurospheres and proliferation when cultured in vitro. Induced by serum medium, they can differentiated into neurons, oligodendrocytes and astrocytes. Tumorigenicity test proved that the cell line without tumorigenicity.(3)RT-PCR showed that the recombinant plasmid transfection of rat fetal NSCs existed the product of hTERT gene mRNA transcripts; Fluorescence microscopy was combined with the Western blot test confirmed that the fusion protein hTERT-GFP expression.(4)Proliferation and cell cycle change at different times of CD133+ cell in Rat fetal NSCs neurospheres, which transferred with recombinant plasmid, were analysed by flow cytometry. This is further confirmed that the hTERT on recombinant plasmid can update the telomerase activity of transferrd NSCs, and also have a role for promoting cell proliferation in vitro.