| Purpose:Cardiac stem cells play a critical role in the maintenance of cardiac tissue homeostasis and the repairing after heart injury.The functional degenerative changes induced by aging may be one of the main causes for age-related heart disease.In this study,we isolate and identify one cardiac stem cell subsets which have differentiation and self-renewal capacity from C57BL/6J mice.Then we elucidate the age-related changes in regard to function of the cardiac stem cell through the analysis and comparison of cell contents,cell cycle,differentiation and self-renewal capacity of cardiac stem cell in youth(2-3 months)and aged(22-24 months)mice.Finally,we analyze the function of Cited 2 and cardiac stem cell gene expression profile between the young and old groups for laying the foundation for exploration about senescence regulation mechanisms.Methods:(1)Isolation of mouse cardiac stem cells:mice were killed by cervical dislocation and then took heart out.Heart single cell suspension was prepared by mechanical shear method,collagenase ? digestion and cell filtration.(2)Analysis and sorting of mouse cardiac stem cells by Flow cytometry:freshly isolated cells were first stained with four kinds of fluorescent-labeled antibodies against relevant surface marker proteins,and then analyzed phenotype,cell cycle and sorted by flow cytometry.(3)Detection the ability of differentiation and proliferation ability in cardiac stem cells:sorted cardiac stem cells cultured in specific differentiation and proliferation medium,then stained with fluorescent-labeled antibodies against relevant marker proteins and CFSE staining for measuring the condition of differentiation and proliferation by fluorescence microscopy and flow cytometry.(4)Research of cited 2 regulation mechanism:constructing cited 2 knockdown and overexpression system by using lentivirus as carrier,sorted cardiac stem cells through lentivirus transfection use its own GFP marker to detect transfection efficiency and select stably transfected cell lines by puromycin after transfection success,then added differentiation medium induced differentiation and detected differentiation.(5)Detection of gene expression:total RNA in isolated cardiac stem cells was extracted,reversed transcribed to cDNA,performed Real-time PCR and ddPCR,and detected the condition of gene expression.(6)Gene expression analysis:GO analysis,KEGG pathway analysis.(7)Statistical methods:T test was applied for all date analysis using SPSS 19.0 statistical software,the data was showed as Mean ± standard deviation.Results:(1)Isolation and identification of mouse cardiac stem cells:Two potential cardiac stem cell subsets in heart tissue of mice by with Sca-1 as the core and combining with three other kinds of surface mark as Lin,CD45 and CD31 were identified:Lin-CD45-Sca-1+CD31-cells and Lin-CD45-Sca-1+CD31+Tcells,which accounted for 0.74±0.03%and 3.38±0.30%.By inducing their differentiation and proliferation,it turned out that Lin-CD45-Sca-1+CD31-cells were able to differentiate into myocardial cells,smooth muscle cells and vascular endothelial cells in vitro,and able to proliferate in vitro,but Lin-CD45-Sca-1+CD31+cells did not possess differentiation and self-renewal capacity,so Lin-CD45-Sca-1 CD31-cells are true cardiac stem cells.The analysis of Micro RNA and mRNA expression spectrum showed that,20 micro RNA and 49 mRNA about stemness in the two subsets of cells present opposite expression,this result adds to evidence that the Lin-CD45-Sca-1+ CD31+cells is the true heart stem cells.(2)Age-related changes in function of mouse cardiac stem cells:content of Lin-CD45-Sca-1+CD31-cardiac stem cells in aged mice was significantly higher than young group(0.92±0.06%VS0.74±0.03%,P<0.05),consistently,heart stem cells in elderly group were also significantly higher than that in the young group in S phase(11.86±2.71%vs 7.72±2.64%,P<0.05).the efficiency of cardiac stem cell in youth group differentiate into myocardial cells(73.40±10.64%vs 40.10±7.84%),smooth muscle cells(81.24±11.23%vs 54.28±9.07%)and endothelial cells(64.85 ± 10.68%vs 37.89±7.47%)was significantly higher than that of the aged group(P<0.01)and proliferation ability is also significantly higher than that of the aged group(72.86±12.62%vs 51.48±9.87%,P<0.05).(3)the regulation of Cited 2 in differentiation of cardiac stem cell:the expression of Cited 2 in cardiac stem cells of old mice was significantly lower than that in the young group(P<0.01).The differentiation ability of Lin-CD45-Sca-1+CD31-cardiac stem cells was slightly decreased and rose after Cited 2 was knockdown and over express.(4)Screening for potential senescent regulation factor in Cardiac stem cell:there were 35 genes existed differential expression between young and aged mice cardiac stem cells(FDR<0.05,fold change?2),10 genes were down-regulated expression and 25 genes were up-regulated expression in elderly group.These genes primarily involved in cell proliferation,cell cycle,rhythm regulation,Transcriptional maladjustment in cancer,chemokine signaling pathway,complement pathway,NF-kappa B signaling pathway and so on.Conclusion:Lin-CD45-Sca-1+CD31-cells are real cardiac stem cells in mice which possess differentiation and self-renewal capacity,the number of Lin-CD45-Sca-1+CD31-cells increased in aged mice,but the differentiation and self-renewal ability decreased significantly.Therefore,the degenerative changes of function in aging heart may mainly be caused by cardiac stem cells function lessening,not cell content reducing.Ancillary transcription factor Cited 2 plays less of a role in differentiation ability regulation of cardiac stem cells,and its main role is to regulate cardiac development rather than cardiac stem cells differentiation process.Aging of Cardiac stem cell is modified strictly by epigenetics,and potential regulatory factor is related to cell self-renewal ability. |
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