| Background:Previous study indicates there are resident stem cells in heart with the ability of self-renewal and multipotent. In2011and2012, two Phase I clinical trials completed using cardiac stem cells for heart disease. Of them, SCIPIO uses c-kit positive cardiac stem cells to treat ischemic cardiomyopathy, which results in significant EF improvement. CADUCEUS uses cardiosphere to treat acute myocardial infraction, which results in regional improvement of cardiac muscle but no significant change of left ventricular ejection fraction. A recent study shows cardiac stem cells are more superior to bone marrow mononuclear cells or mesenchymal stem cells of therapeutic effect on acute myocardial infraction in mice. However, the isolation and expansion of cardiac stem cells more sophisticate. Therefore, establish an effective method is essential for future basic and clinical research. At the same time, lessons from previous studies told us that the effect of using stem cells alone to treat heart failure is limited because of the low engraftment and retention rate and harmful microenvironment. So current research focuses on methods to improve the violability of stem cells and microenvironment.Objective:1) Establish the method to isolate and expansion of c-kit positive cardiac stem cells from adult heart tissue.2) Explore the effect of atorvastatin on cardiac stem cells in hypoxia and serum deprivation injury.Methods:1)The isolation and expansion of cardiac stem cells:the hearts are obtained from adult C57BL/6mice, then minced and digested with0.2%trypsin and0.1%collagenase IV. After digestion, the cardiac explants are cultured in complete explants medium (CEM) coated with matrigel. After about two weeks of growth, we collect the small, round, phase-bright cells emerged on the layer of fibroblast-like cells. These cells are further cultured in cardiosphere growing medium (CGM) coated with poly-D-lysine. After expansion, we purify these cells using magnetic-activated cell sorting (MACS) with c-kit magnetic beads. Flow Cytometry (FCM) are then preformed to examine the expression of stem cell surface markers including c-kit and sca-1antigen.2) The effect of atorvastatinan cardiac stem cells in hypoxia and serum deprivation (H/SD) injury:the sorted cardiac stem cells are culture for12hrs in H/SD with different gradients of atorvastatin (control,10-3μmol/l,10-2μmol/l,10-1μ mol/l,1μmol/l,10μmol/l). Cells morphology are evaluated using phase contrast microscopy and the rate of apoptosis are measured with FCM.Results:1) The isolation and expansion of cardiac stem cells:After about three days of culture, fibroblast-like cells emerge from adherent cardiac explants, about one week later, some small, round and phase-bright cells start to migrate. After collection and expansion, inverted phase contrast microscope examination shows relative uniform spindle-like cells with several clone-like proliferation. We could get about1%cells after magnetic-activated cell sorting (MACS). The purity of sorted c-kit positive cells are characterized by flow cytometric analyses. Before magnetic separation,3.44%cells are positive for c-kit expression,53.46%for sca-1expression, while18.59%are positive for c-kit expression after MACS.2) The effect of atorvastatinon cardiac stem cells in hypoxia and serum deprivation (H/SD) injury:For high concentration atorvastatin groups, the cells show more shrink by microscopy and greater rate of apoptosis rate by FCM compared with control group (control:20.5%,1μ mol/l:29.5%,10μ mol/l:28.2%, p<0.05). As for low concentration atorvastatin groups, the cells show no significant difference compared with control group (control:20.5%,10-3μ mol/l:19.2%,10-2μ mol/l:23.2%).Conclusion:c-kit positive cells could be obtained from adult mouse heart tissue using enzymatic dissociation method and purified with magnetic-activated cell sorting. The effect of atorvastatin on cardiac stem cells needs further investigation. |
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