| Stem cells are cells haveing the ability to self-renew and differentiate into a variety of specific types of cells.,including Embryonic Stem Cells,(ESCs),Embryonic Germ Cells,(EGCs)and induced Pluripotent Stem Cells(iPSCs)etc.Induced pluripotent stem cells are pluripotent stem cell lines with ES cell morphology and growth state and expressing ES cell markers obtained by introducing pluripotent four factors into somatic cells and culturing in ES medium.Compared with ESCs and EGCs,iPSCs have similar pluripotency to them and can be directed to differentiate into many types of tissue cells.At the same time,iPSCs have a wide range of sources of donor cells,universal applicability of technology,and the ability to avoid social ethical issues.It has extremely important academic value and broad application prospects in major disease cell therapy,drug screening,disease diagnosis and treatment,regenerative medicine,animal genetic resources protection and establishment of transgenic animal models.Pigs,as a common experimental animal model,have gradually become an ideal animal model for human xenotransplantation,cell therapy and regenerative medicine because of their high similarity with humans in terms of immunology,histological morphology and physiological structure.In particular,the human organelles produce specific organ tissue chimeras in pigs through xenogeneic embryo transplantation,thereby providing humans with a wider range of organ and organ transplantation donors.Good pre-application and wide-ranging requirements put forward urgent expectations for the establishment of porcine ES cells and pig iPS cells.In this experiment,we first carried out mRNA-Sequence analysis of normal cultured pigs Dox-iPS,Feeder-Dox-iPS,and Feeder-Dox-iPS,and found that the initiation of pluripotency four-factors at the same time as initiating cell reprogramming,Endogenous Lin28 a expression was significantly up-regulated.On this basis,we constructed a double luciferase reporter vector in the promoter region of Lin28 a and hybridized with dual luciferase with a pluripotency fourfactor vector and found that Sox2 can specifically bind to the Lin28 a promoter region and upregulate Lin28 a expression.To verify the initiation of Lin28 a in the reprogramming process,we isolated the primary porcine Sertoli cells as donor cells for four-factor induction,picked the monoclonals on the 15 th day of induction,and obtained three DOX-iPS cell lines,which were observed by morphology and immunofluorescence staining.And endogenous pluripotent gene detection revealed that the reprogramming process of porcine supporting cells was accompanied by the activation of Lin28 a.We isolated pig support cells,induced four-factor reprogramming,induced monoclonal picking on 15 days,and obtained three DOX-iPS cell strains.The pigs were supported by morphological observation,immunofluorescence staining,and endogenous pluripotent gene detection.Cell reprogramming is accompanied by the activation of Lin28 a.To further determine the function of Lin28 a in porcine DOX-iPS cells,we constructed CMV-Lin28a-MCS-EF1-copGFP,TRIP-EF1-Lin28 a,TRIP-EF1-GFP and Lin28 a interference vectors and obtained DOX-Lin28a-In the iPS and DOX-shLin28a-iPS cell lines,it was found by quantitative detection that Lin28 a was overexpressed in DOX-iPS cells,and the pluripotent gene(Sox2,etc.)did not significantly change,but the cell proliferation was accelerated,the cell clone shape became flat and interfered with Lin28 a.Afterwards,cell proliferation slowed down,but intracellular pluripotency genes such as Oct4 were significantly upregulated,indicating that during the reprogramming process,the regulation of Lin28 a by Sox2 and Oct4 antagonized each other,thus realizing the transition from the Prime to the Naive state of reprogramming.Our experiments proved that Lin28 a is a key node in the maintenance of pluripotency in pig iPS cells.Up-regulation of Lin28 a promotes the proliferation of pig iPS cells,and in prime state iPS cells,the expression level of pluripotency-related genes such as Oct4 increases after interfering with Lin28 a cells,and pluripotency has been improved. |
