The Study Of The Function Of Ctgf In Hescs Proliferation And Differentiation Into Definitive Endoderm

Objective:Study the function of connective tissue growth factor in hESCs proliferation and differentiation into definitive endoderm, and clear its regulation mechanism.Methods:First, hESCs were cultured on mouse embryonic fibroblasts feeder cells and matrigel, Then detected the expression levels of marker gene of hESCs by immunofluorescence and alkaline phosphatase staining, thus proved the differentiation pluripotency of the cells. On this basis, induced the hESCs differentiation into DE by the combination factors of ActivinA and Wnt3a, and the differentiation efficiency were detected by flow cytometry.On the basis of the above work, we launched a study of CTGF on the proliferation and differentiation of human embryonic stem cells. On the one hand, we investigated the function of CTGF on hESCs proliferation by the method of alkaline phosphatase staining; On the other hand, the role of CTGF in the differentiation to DE cells of hESCs was analysised by flow cytometry.To study the mechanism of CTGF in hESCs proliferation and differentiation further, we detected the role CTGF play on phosphorylation of protein in MAPK signaling pathway and TGF-β signaling pathway by Western blot.Thus identified the signaling pathway of CTGF regulation initially.At last, we combined the miRNA microarray screening results which get from our laboratory’s previous work,studied the regulation of several miRNAs on CTGF which target to CTGF and expressed differentially after the hESCs differentiation.These several miRNAs’ regulation effects on CTGF expression were tested by real-time quantitative PCR and Western Blot, the results showed that miR-375have a most obvious regulation on CTGF.The next, we predicted the binding sites of miR-375and CTGF by bioinformatics analysis software TargetScan and PicTar,and clear their combination relationship by Luciferase eventually.Finally,we studied the effect of miR-375on hESCs proliferation by alkaline phosphatase staining.Results:Immunofluorescent staining and alkaline phosphatase staining results showed that the Oct4, Nanog, and alkaline phosphatase were expressed at high levels in our hESCs, indicated that the hESCs have a good differentiation potential.After induced the hESCs differentiation to DE cells, the Sox17expression levels was high,and the flow cytometry results showed that the CXCR4-positive cells is more than50%,indicating that we have established a successful differentiation model of hESCs to DE cell.In the study of the role CTGF play in hESCs proliferation and differentiation to DE, Western Blot results showed that the expression of CTGF in the process of hESCs differentiation to DE cell was lowered significantly.In addition, we also found that the CTGF has a role in promoting the hESCs proliferation,and have a opposite effect on hESCs differentiation to DE. Our study found that CTGF can promote the MAPK signaling pathway and TGF-β signaling pathway in hESCs.In the research of CTGF expression, we found that miR-375have a most obvious effect on CTGF regulation. The Luciferase results showed that miR-375play a regulatory role through combinating with CTGF3’UTR region. In addition, we also found that overexpressed miR-375can inhibit the proliferation of hESCs.Conclusion:1、CTGF has a obvious effect on promoting hESCs proliferation and inhibiting the differentiation from hESCs to DE.2、The regulatory role of CTGF play on hESCs proliferation and differentiation to DE cells may be associated with MAPK and TGF-β signaling pathway in hESCs.3、MiR-375regulated CTGF expression by combinating with CTGF3’UTR region.Meaning:Functional cells of the liver and pancreatic were developed from endoderm cells, therefore inducing hESCs differentiation to DE is the prerequisite for getting mature liver and pancreatic cells,is the basis for human tissue repair and organ regeneration,and is the hope for a radical cure of severe liver disease and diabetes ultimately. Therefore, the study of the mechanism of hESCs maintain and differentiation to DE cells have a very important significance.Our subject research focus on the function of CTGF in hESCs proliferation and differentiation to DE cells, it is a part of the study of hESCs maintaince and differentiation, It has a great significance for people to recognize the mechanism of directing hESCs to mature cells,and also has potential applications for the treatment of diseases.