Experimental Study On Differentiation And Proliferation In BMSCs-derived Neural Stem Cell By Transcription Factor Hes5

Hes5 gene, a key downstream mediators of Notch signaling that functions by inhibiting the expression or activity of positively acting bHLH factors, is a member of bHLH transcription factor family which acts as a positive-negative transcriptional regulators which is expressed transiently in different stages in cells growth and differentiation and orchestrate those precisely timed and progressive maturational events underlying cell fate decisions according to orders of time and spatial and coordinated the function of majority of signaling transduction on target genes to influence the proliferation and differentiation. bHLH and WRPW domain in c-domain which are peculiar to Hes5 endowed them the ability to inhibit the transcription by combined with the starter of achaete-Scut and MyoD, which promote the gene differentiation. It is deemed that the acting of Hes5 is able to maintain neural stem cell stable and reduces the differentiation. However, it is not quite distinct that whether Hes5 expressed in BMSCs-derived neural stem cells (NSCs), what are its functions, and what might be influence on NSCs caused by breaking the expression of it.In order to find a resolution to the problem, a series of studies have been conducted concentrating on the function of Hes5 in BMSCs-derived NSC. Inthe first places, the characteristic of growth of BMSCs and its ability to form neural cells based on the previous laboratory condition has been observed and an initial study in mechanism has also been carried out in order to found a standard and ideal culture system; Secondly, it has been checked whether and when BMSCs-derived NSCs express Hes5 and it has also been analyzed whether the small double-strain RNA from artificial manufacture can inhibit the endogenesis Hes5 and establish the best dsRNA concentration for transfection, either keep the considerable amount cell vigor or break the expression of endogenesis Hes5 ;Lastly it is discussed the influence on NSCs differention and proliferation by blocking the Hes5 expression.By statistical analyses of the detected indices, it can be revealed that BMSCs can form cell group like island express Nestin in vitro in special NSCs culture medium and differentiate to cells which exist in nerve system; Meanwhile it is suggested that bFGF and EGF can promote the BMSCs proliferation and shorten population doubling time and BDNF and RA is able to increase the expression of the special antigen of mature nerve cell and glia cell; it is found through the experiment that BMSCs began to express Hes5 at 5th day after cultured in vitro and the expression increased progressively with time pass; dsRNA at 200nmol/L can inhibit the expression of Hes5 peculiarly and is favorable to the living of cells; statistic comparison between transfected group and non-transfected showed that there is significant difference in the expression of NeuN and GFAP; cells differentiation mainly express GAD and result in the increase of the quantity of Mash 1,a downstream gene of Hes5, no obvious positive changes in PI6 and P27.From the experiment, it can be concluded that NSCs derived from bone marrow of mammal can be conversed into nerve cell and glia cell if there is suitable culture condition and suitable concentration of cytokines and these cells keep the ability of proliferation and differentiation. And formed cells partly express GFAP and others express NSE positive, it can also be deducedthat Hes5 is beneficial to the conversion of BMSCs to NSCs and keep the proliferation and dramatically reduce the differentiation to the next step; exogenesis dsRNA is a new and effective gene-study tool because it can degradate the homologous mRNA transiently and effectively; mainly, the control of gene expression is positive and maybe another factors take part in this process; signaling transduction in cell are very complexity and refinement which is liable to be interfered when another conducting passage is being investigated.