The Function And Mechanism Of Lgr5 In Cell Reprogramming

Objective: 1.To determine the effect of adult stem cell marker Lgr5 on cell reprogramming.2.To explor the presence of a suitable ligand for Lgr5 involved in cell reprogramming.3.To explore the mechanism of Lgr5 affecting cell reprogramming.4.To solve the biomedical problems using the effect of Lgr5 on cell reprogamming.Methods: First,the expression of Lgr5 in multiple mouse tissues was detected by q PCR.The overexpression vector or knockdown vector was selectively constructed according to the expression level.We constructed the overexpression vector on p FUW-tet-on vector whose expression was controlled by Doxycycline(Dox),and packaged viruses using 293 T cells and ps PAX2 and pmd2.G virus packaging plasmids.To detecting the effects of Lgr5 overexpression on mouse embryonic pluripotent stem cells(m ESCs),we directly infected m ESCs with viruses or established stable mouse embryonic stem cell lines of Lgr5 overexpression.The same strategy was used on detecting the effects of Lgr5 knockdown.For the effects on m ESCs,we mainly observed the cell morphologies and tested the expression of the pluripotent marker genes such as Nanog,Oct4,Rex1,Fgf5 and so on.Mouse embryonic fibroblasts(MEFs)were used as the somatic reprogramming starting cells to explore Lgr5's influence on cell reprogramming.Using the inducing system,which had been successfully established,we obtained MEFs capable of direct expression of Oct4,Sox2,Klf4 and c-Myc by adding tetracycline to induce cell reprogramming.The overexpression or knockdown of Lgr5 in this cell was carried out by viruses infection.We compared the colony formation rate and efficiency,and dated the colony formation.In addtion,we screened some proteins associated with Lgr5.Wnt signaling pathway played an important role in maintenance of cell stemness.Lgr5 as a G protein coupled receptor,was involved in Wnt signaling pathway.Rspo3 as the homologous ligand of Lgr5,participated in Wnt signaling pathway in conjunction with Lgr5.The same method was applied to detect the effect of Rspo3 overexpression on reprogramming efficiency and the stemness of m ESCs.We detected the expression change of ?-catenin –the core factor of Wnt pathway—at the RNA and protein level by Real-time q PCR and western blot.Ch IP-seq was used to analyse the DNA sequences binded by key transcription factors in different types of cells and the downstream target genes of Lgr5 and Rspo3,then to reveal the mechanism of Lgr5 in cell reprogramming.Results: 1.The overexpression of Rspo3 could promote the reprogramming effeiciency of MEFs.2.The expression of Lgr5 and Rspo3 in m ESCs could up-regulate the expression levels of the pluripotent genes.3.The overexpression of Lgr5 in m ESCs increased the expression level of Wnt pathway core protein--?-catenin.Conclusion: Lgr5 and Rspo3 overexpression synergistically promoted the reprogramming of mouse embryonic fibroblasts and maintained pluripotency of mouse embryonic stem cells.