| Neural stem cells (NSCs) have become currently a hot point of neuroscienceresearches, because it has self-renewal ability and multi-directional differentiationpotency. But researches of NSCs are still in exploratory state, the culture anddirectional differentiation of NSCs have difficulties. NSCs can be gotten fromembryos and adult brains of animals including the human, and maintain potenciesof self-renewal and multi-directional differentiation for long-term in vitro asmitogen exist. The survival of NSCs requires glucose, and lack of glucose maylead to the loss of functions and NSCs dying. Glucose is one of pacing factor ofculturing NSCs in vitro.Culturing NSCs in vitro is the precondition of researching the mechanisms ofproliferation and differentiation of NSCs, and that the mechanisms of proliferationand differentiation of NSCs are deeply known is the key of clinic application ofNSCs. At present, it is greatly thought that proliferation and differentiation ofNSCs are mainly regulated by microenvironment and genes. researches find that bHLH genes play a key role in the development, proliferationand differentiation of many tissues and cells, especially central neural system(CNS). There are two types of bHLH genes, the repressor type including Hes andthe activator type including mash1and math. Two types of bHLH genes retaindynamic balance and regulate and determinate differentiation and fate of NSCstogether. Our experiments will explore primary culture and mechanism ofproliferation and differentiation of NSCs, respectively.Part I Primary culture of NSCs from mouse fetal brain in vitro and influenceof glucose concentration on its proliferation and differentiation capability.1. Primary culture and identification of NSCs from mouse fetal brain in vitro.The embryo-brain of Kunming mouse of E13 or E14 was dissected and separated.Then the dissociated cells were cultured in medium containing EGF and bFGFand passaged in time. The cells suspended formed neurospheres in the medium. Toidentify the cells immunocytochemistry and immunofluorescence stain was used.It was found that the neurospheres showed a positive reaction to nestin antibody,which illustrated the cells were undifferentiated. The neurospheres passaged threetimes were transferred into the medium containing 10% fetal bovine serum but noEGF and bFGF. The neuropheres gradually adhered to the bottom and some cellsemigrated from the rim of the neuropheres and differentiated into neuron-like orglial-like cells in the medium containing serum. The differentiated cells showed apositive reaction to Neurofilament200(NF200) or glial fibrillary acidic protein(GFAP) antibody, which illustrated that the differentiated cells were neuron andglial. In a word, we got NSCs through culture in vitro. NSCs. NSCs passaged three times were transferred into noraml physiologicalglucose concentration (PG) and high glucose concentration (HG) mediumcontaining EGF and bFGF, respectively. Then MTT assay was used to draw thegrowth curve of the cells in PG and HG medium. The results found that NSCs hadhigher proliferation ability in PG medium than in HG medium. To examine theeffect of glucose concentration in the differentiation of NSCs, NSCs cultured inPG and HG medium were transferred into PG and HG medium containing 10%fetal bovine serum but no EGF and bFGF, respectively, to differentiate for 3d and10d. Immunofluoresence stain of NF-200, GFAP and nestin was used to identifythe differentiated cells. It was found that there was higher positive proportion ofNF-200 and GFAP and lower positive proportion of Nestin in HG medium than inPG medium, after NSCs were induced to differentiate for 3d; to continue todifferentiate for 10d, nestin positive cells were not observed in tow groups, whichillustrated that NSCs had already differentiated completely. Immunofluorescencestain of NF-200 was used to identify the differentiated cells. The results showedthat there was the higher positive rate of NF-200 in PG medium than in HGmedium. Therefore, we concluded that HG impaired the proliferation capacity ofNSCs , accelerated the early differentiation of NSCs and finally, decreased thedifferentiation rate of neuron.Part II Expression of relative bHLH genes in the differentiation of NSCsNSCs passaged in vitro were transferred into gelatinum-coated 6–well platesand induced to differentiate with all-trans retinoic acid. reverse transcriptionpolymerase chain reacti(RT-PCR)detected expression of relative bHLH genes: hes1, hes5 and mash1, math1, NeuroD in undifferentiated and differentiated cellsfor 1d, 2d, 3d, 5d and 10d. The results showed that Hes1 gene was expressed inundifferentiated and differentiated cells; expression of Hes5 gene decreased withtime; expression of NeuroD and Mash1 genes was only obvious in differentiatedcells; expression of NeuroD gene was evident for 2 RA days, and reached thepeak in the intermediate stage of differentiation; expression of Math1 gene wasonly relatively evident in late stage of differentiation. The results showed thatexpression of the repressor type bHLH genes was gradually dreasing, butexpression of the activator type bHLH genes was gradually increasing in thedifferentiation of NSCs. However, for each bHLH gene, even bHLH genes of thesame type, expression of them were very different. Meanwhile, combined togetherwith departed reseach: applying anti-Hes1 to block expression of Hes1 in NSCsmay induece NSCs to differentiate into GABAergic neurons. We thought that thepattern of expression of bHLH genes played a key role in the differentiation.Regulation of differentiation of NSCs may be achived by changing the pattern ofexpression of bHLH genes.
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