Biological Characterization Of Cardiac Progenitor Cells Drived From Embryonic Heart And Preliminary Study On Islet1 Gene

To clarify the biological characteristics of the chicken embryonic cardiac progenitor cells, we made a study on isolation, isolated culture, identification, differentiation, biological characteristics, Self-differentiation of the ECPCs and we also made a preliminary exploration of the chicken gene Islet1. We got the following results:1. The test compared two isolation methods: tissue culture and enzyme digestion culture method. Finally, we made a selection for enzyme digestion culture as a conventional isolation method for chicken cardiac progenitor cells. The chicken cardiac progenitor cells obtained by Enzyme digestion can be expanded stably in vitro, and sub-cultured to 18th passage.2. We explored the chick hatching time for experiment and found that the chick embryos amoung 6d-11d were suited for operation, and could got a large number of relatively pure target cells.3. Different culture conditions on proliferation of chick embryo cardiac progenitor cells have greater impact: chicken cardiac progenitor cells are very sensitive to pH in culture medium, the dramatic changes of PH will lead to cell apoptosis; chicken embryonic cardiac progenitor cells have a serious selection for the conditions of the culture medium, they could proliferated rapidly while we used the ECPCs DMEM/F12 proliferation medium.4. In morphology, the chick embro cardiac progenitor cells showed a faster, clonal proliferation, morphology with oval, polygonal, a high ratio on nucleus and cytoplasm. We can observe a three-dimensional-like structure of cells clearly by Phase contrast microscope.5. We made immunohistochemical and RT-PCR identification on the 3rd and 11th passsge of chicken cardiac progenitor cells and the results showed that chicken cardiac progenitor cells can express Isl1, GATA4, Flk1, and Nkx2.5.6. Chicken cardiac progenitor cells can differentiate into mature cardiac cells and smooth muscle cells in appropriate inducing conditions, the immunohistochemical and molecular identification results of the induction showed that: The rhythmic beat contraction of myocardial cells occurred after induced, cTnT expression was positive; theα-SMA expression of smooth muscle cells after induced was positive.7. The CDS region and LIM homeodomain fragment of Islet1 gene was successfully cloned, and the eukaryotic expression vectors were completed.8. The bioinformatics analysis of Islet1 protein sequence showed that Islet1 protein has three structure domains and high homology in different species.