| BackgroundAdministration of different types of stem cells has been considered in preclinical and clinical research of MI. Cardiac stem cells (CSCs) are shown to be self-renewing, clonogenic, and multipotent, exhibiting the ability to differentiate into cardiomyocytes, smooth muscle cells and endothelial cells both in vitro and vivo. For this reason, cardiac stem cells have sparked intense interest. Growth factors can strongly mobilize stem cells and increase the adhesion and proliferation force of stem cells, The mechanism of growth factors in repairing heart damage is complex, hepatocyte growth factor (HGF) and insulin-like growth factor-1(IGF1) can induce a variety of stem cells into cardiac cells, but its effect on cardiac stem cells are rarely reported. In this study we examined the effect of GF in stem cells.ObjectiveTo investigate the induction effects of HGF and IGF1on CSCs, observe CSCs proliferation and directly differentiation into cardiomyocytes. Provide a potential theoretical basis for CSCs clinical application of myocardial infarction.MethodThe myocardial tissues were dissected for primary culture of CSCs by methods of explants and primary culture of cardiomyocytes by methods of enzymatic digestion. Immunofluorescence techniques to identify cardiac stem cell specific surface marker c-kit and blood-derived stem cell surface marker CD34, sorting c-kit+cardiac stem cells by flow cytometry, CFDA SE green fluorescent tracer probe to detect the proliferation of cardiac stem cells. Primary cells were separated and purified by flow cytometry. The proliferation characteristic of c-kit+CSCs was detected with CFDA SE fluorescent probe. c-kit+CSCs were divided into two groups. One group was only intervened with growth factor; another group was co-cultured with cardiomyocytes and intervened with growth factor. Two weeks later, the cell count and morphological changes were observed with living cells workstation. The Nkx2.5and cardiac troponin T were detected by immunofluorescence.Results1.Simple cardiac stem cell groups:inverted microscope cardiac stem cell number and morphology did not change significantly, remains small round and light patterns; cultured for5days, in control group cardiac stem cells floated and dead. The other three groups had no such phenomenon, the growth of the normal state. To observe by living cells workstation CFDA SE fluorescence intensity of cardiac stem cells, no changes in the strength, CSCs did not proliferate.2. Cardiac stem cells co-cultured with cardiomyocytes groups:In each group, CSCs proliferated and occurred morphological changes, under inverted microscope, cardiac stem cells show short spindle, or relatively large round; under living cells Workstation, CSCs CFDA SE fluorescence intensity was weak, cardiac stem cells proliferated; Nkx2.5and cTnT of cardiac stem cells expressed, in HGF+IGF1group, cardiac stem cells differentiated into spontaneously beating cardiomyocytes.Conclusions1.Growth factors can prolong the survival time of cardiac stem cells, but had no effect on proliferation and differentiation of cardiac stem cells;2.Co-cultured with cardiomyocytes, hepatocyte growth factor and insulin-like growth factor could induce cardiac stem cell to proliferate and differentiate into cardiomyocytes, but spontaneously beating cardiomyocytes induction rate is relatively low. |
