Immortalization Of Embryonic Neuarl Progeniter Cells And Identification Of Its Characters And Functions

Objective:The central nervous system regeneration is difficult due to nerve regeneration of adverse environmental factors, and is generally thought that neuronal cells can’t to carry on the division and proliferation but are terminal differentiation cells. Neural stem cells have self-renewal and replicate division abilities, which was supposed to replace and treat various diseases of the nervous system, a series of their potential value was discovered only by recently, neural stem cells with high mobility and can be attracted and moved to the lesion side such as in brain ischemia and pathological changes by the tumor, thus it become a very promising tool for clinical therapy.This study intends to use retroviruses to exogenously carry SV40T Ag gene flanked with LoxP sites into embryonic neural stem cells to immortalize the mice embryonic neural stem/progenitor cell line, Use specific markers to identify immortalized neural stem/progenitor cells phenotype, and the possibility of potential multiply differentiation. In addition, use Cre recombinase knockout SV40-T to reverse immortalized neural stem cells and change its proliferation characteristics, making it back to primary cells and has the ability of to differentiate into different neural cells. In vivo studies confirmed that immortalized neural progenitor cells were distinguished from tumor cells, would not be aggressive. The study shows that the iNPC has the value of wide range clinical and research use, which provides a new method for regeneration and functional recovery of central nervous system.Methods:Part1:Pregnant12.5days and14days CD1mice each, obtained the12.5days and14days embryo from uterus of the pregnant mice. Dissected telencephalon of fetal mouse brain tissue under dissecting microscope. Single cell suspension were made by the pancreatic enzyme digestion, then using the neurosphere method to culture neural stem cells. After neuroshperes forming for3days, The neural stem cells were mechanically isolated to single cell and attached to the dishes. At the same time, the expressed plasmid SSR#69containing SV40T antigen and retroviral skeleton plasmid pCL-Ampho were co-transfected to HEK293cells.293cell packaged retrovirus containing SV40T antigen gene and secreted retrovirus into the culture medium, then the retrovirus supernant was used to infected the attached neural progenitor cells. Through pressure screening by hygromycin B antibiotics, positive clones which were immortalized neural progenitor cells were obtained and expanded in the culture. Part2:Immortalized neural progenitor cells (iNPCs) were passaged for several generations before appraisal, RT-PCR was used to assess iNPCs self-renewal ability and neural cells specificity, and its representation of neural progenitor cells of different brain regions, immunofluorescence staining was further used for identification of potential of differentiating into different neural cell lineages. Recombinant adenovirus Ad-Cre was used to infect iNPCs, SV40T was knock out to get reversed immortalized neural progenitor cells (NPCs), set Ad-GFP infected iNPCs group as control group. Through trypan blue staining, MTT test, crystal violet staining and cell cycle analysis, proliferation ability of iNPC and NPC were evaluated. In vitro iNPC and NPCS differentiation were induced by differentiating conditions, used RT-PCR, immunofluorescence staining to identify iNPC and NPC differentiation to mature neural cells. Finally the membrane potential of iNPCs function were analyzed. And in vivo transplantation with iNPC and NPC to evaluate the proliferation ability.Results:Part1:E12.5and E14embryonic neural stem cells were successfully obtained, Abundant of neurospheres were formed during culture, neurospheres were mechanically separated to single neural stem cells and attached to the dishes. SV40T gene was retrovirally integrated into the embryonic neural progenitor cells genome and successfully obtained immortalized neural progenitor cell line, which harbored the abilities of constant proliferation and division. Part2:By RT-PCR, we proved that the immortalized cells were neural progenitor cells, and specific source of the central nervous system cells. iNPC could represent the forebrain, midbrain, the ventral brain areas, etc. And iNPC has the potential to differentiate into neurons, astrocytes and oligodendrocytes. Immunofluorescence staining confirmed that iNPC widely expressed NPC markers nestin and beta3-tubulin antigens. After Ad-Cre and Ad-GFP infection, the morphology of Ad-Cre group iNPC cells gradually changed to form primitive cells, but the Ad-GFP group cells kept their original form. Cell proliferation test found that Ad-GFP group cells has obvious proliferation capacity, and Ad-Cre group cells tend not to proliferation and dieing in the process of cultivating, indicated that Cre recombinase knocked out SV40T from iNPC. Flow cytometry analysis found that Ad-GFP group mainly is in G2phase, but the Ad-Cre group cells mainly stayed in G1phase. Immunofluorescence staining found partial of reversely immortalized iNPCs were MAP2, GFAP and04positive cells, suggested their multipotent differentiation capacity. Cell membrane potential analysis found iNPCs could be induced some immature action potential. In vivo transplantation iNPC and NPC found no proliferation cells in ectopic tissues.Conclusion:This study successfully established immortalized neural progenitor cell line iNPC, experiment proves the possibility of unlimited proliferation in vitro is not limited by culture conditions. Proved immortalized neural progenitor cells could represent different brain regions, and multipotent of differentiation. The immortalization is reversible, iNPC could be reversed as primary cell under certain conditions and changed its proliferation characteristics. In vivo study showed iNPCs were noninvasive, is different from the tumor cells. The established iNPC has extensive clinical and research applications.