The Effect Of AngⅡ And 5-aza Induce Mice Adipose-derived Mesenchymal Stem Cells Differentiation Into Cardiomyocyte Cells And Endothelial Cells

Background and objectiveIt is difficult to restore heart function by myocardial regeneration,if myocardial infarction occurred. Adipose-derived mesenchymalstem cells became to the seed cell of Cellular cardiomyoplasty (CCM),because of its special advantages. At present, the research of ADMSCS is rare reported that ADMSCS become divided into Myocardial vascular endothelial-like cells.The objective of this research is observing Ang Ⅱ combine with 5-aza work on Adipose-derived mesenchymal stem cells of mice induced into cardiomyocyte-like cells,at the same time we observe the effection of it become divided into endothelial-like cells. We want to find some information to support research and theory basis for treat ischemic heart disease in clinic.MethodsLADMSCs’isolation and culture in vitro:After preparation of mice fat tissue which from groin, we use differential attachment method to purify cells in vitro and detected. Observation the morphology character and biological behaviour of mice ADMSCs,use the MTT to draw the growth curve, use immuno-fluorescence to test surface antigenCD90, CD105, CD73, CD45.2.The experiment groups:(1) Contrast group:using the ADMSCs were grown in complete medium.(2) 5-aza group:adding the medium which contents 5-aza to induce for 24h, then change into the complete medium.(3) Ang Ⅱ group:adding the medium which contents Ang Ⅱ to induce for 24h, then change into the complete medium.(4) the combined induction group:adding the 5-aza and Ang Ⅱ to induce for 24h, then change into the complete medium.3.Observation the dynamic growth and the morphology character of cells which are been induced by the inverted phase microscope;Making use of the immuno-fluorescence to identify the expression of CD31 and cTnT and make use of the western blot to test the contents of the protein of cTnT.Results1.Morphological characteristics of ADMSCs cultured in vitro:Under the microscope we found that the nucleus of the original ADMSCs had the smaller vollme, and the shape of it varies from spindle, ellipse to irregularity, extruding with the’ different lengths. The third generation cells have the bigger volume,which has the colony growth and the uniform shape, with a regular arrangement. After induced,cellular morghology induced by 5-aza becomes diversification, which has short rod and irregularity. Some of cellular morghology have unsharp cell membrane and cytoplast with a cavity. Cells that are induced by Ang Ⅱ group and the combined induction group have faster growth rates, uniform cellular morghology, sharp cell membrane, cytoplast with no cavity, major matrixes, overlapping growth, gathing like groups and some of which have a circular distribution.2.The result of MTT:The growth curve of the ADMSCs presented the shape of "S", the first to the third days were the cell latency, and the fourth day was the growing period, when came to the seventh day, it is the peak period.3.The immuno-fluorescence detection results showed that the surface of the CD90,CD105,CD73 was positive, and the surface of CD45 was negative. Ang II group and the combined induction group of 5-aza and Ang Ⅱ could express the CD31.Except the contrast group,all of the other groups could express the cTnT.4.The results of western blotting showed that5-aza group, Ang Ⅱ group and the combined induction group of 5-aza and Ang Ⅱcould express cTnT, and the protein in cTnTof the combined induction group is the highest in all groups.Conclusion1.5-aza was combined with AngⅡ can induce ADMSCs to become into cardiomyocyte cells, which combined with Ang Ⅱ can enhance the differentiation rate of ADMSCs.2.AngⅡ can induce ADMSCs to become into endothelial cells and promote cell proliferation.