解剖学报
解剖學報
해부학보
ACTA ANATOMICA SINICA
2014年
5期
663-669
,共7页
心肌干细胞%增殖%心肌分化%肝细胞生长因子%类胰岛素样生长因子%免疫荧光%大鼠
心肌榦細胞%增殖%心肌分化%肝細胞生長因子%類胰島素樣生長因子%免疫熒光%大鼠
심기간세포%증식%심기분화%간세포생장인자%류이도소양생장인자%면역형광%대서
Cardiac stem cell%Proliferation%Cardiac differentiation%Hepatocyte growth factor%Insulin-like growth factor%Immunofluorescence%Rat
目的：探讨肝细胞生长因子（HGF）和类胰岛素样生长因子（IGF1）在外能否诱导心肌干细胞（CSCs）发生增殖并向心肌细胞定向分化。方法组织贴块法分离培养心肌干细胞，免疫荧光技术鉴定c-kit和CD34表达，流式细胞仪分选纯化c-kit ＋细胞，CFDA SE荧光探针示踪培养检测细胞增殖特征。实验分为单纯心肌干细胞组和与心肌共培养组，分别用HGF和IGF1干预，倒置显微镜观察细胞不同时期数量与形态的变化，活细胞工作站观察CFDA SE示踪剂荧光强弱，采集图像，进行统计学分析。免疫荧光技术检测Nkx2．5、心肌肌钙蛋白T（ cTnT）的表达。结果单纯心肌干细胞组，生长因子刺激后心肌干细胞数量与形态均无明显变化。与心肌共培养组，细胞均发生增殖与形态变化，Nkx2．5、cTnT阳性表达，有个别心肌干细胞分化为自发搏动的心肌细胞。结论心肌干细胞与心肌细胞共培养条件下，HGF和IGF1能够促进心肌干细胞增殖，联合作用能够诱导心肌干细胞向心肌细胞分化。
目的：探討肝細胞生長因子（HGF）和類胰島素樣生長因子（IGF1）在外能否誘導心肌榦細胞（CSCs）髮生增殖併嚮心肌細胞定嚮分化。方法組織貼塊法分離培養心肌榦細胞，免疫熒光技術鑒定c-kit和CD34錶達，流式細胞儀分選純化c-kit ＋細胞，CFDA SE熒光探針示蹤培養檢測細胞增殖特徵。實驗分為單純心肌榦細胞組和與心肌共培養組，分彆用HGF和IGF1榦預，倒置顯微鏡觀察細胞不同時期數量與形態的變化，活細胞工作站觀察CFDA SE示蹤劑熒光彊弱，採集圖像，進行統計學分析。免疫熒光技術檢測Nkx2．5、心肌肌鈣蛋白T（ cTnT）的錶達。結果單純心肌榦細胞組，生長因子刺激後心肌榦細胞數量與形態均無明顯變化。與心肌共培養組，細胞均髮生增殖與形態變化，Nkx2．5、cTnT暘性錶達，有箇彆心肌榦細胞分化為自髮搏動的心肌細胞。結論心肌榦細胞與心肌細胞共培養條件下，HGF和IGF1能夠促進心肌榦細胞增殖，聯閤作用能夠誘導心肌榦細胞嚮心肌細胞分化。
목적：탐토간세포생장인자（HGF）화류이도소양생장인자（IGF1）재외능부유도심기간세포（CSCs）발생증식병향심기세포정향분화。방법조직첩괴법분리배양심기간세포，면역형광기술감정c-kit화CD34표체，류식세포의분선순화c-kit ＋세포，CFDA SE형광탐침시종배양검측세포증식특정。실험분위단순심기간세포조화여심기공배양조，분별용HGF화IGF1간예，도치현미경관찰세포불동시기수량여형태적변화，활세포공작참관찰CFDA SE시종제형광강약，채집도상，진행통계학분석。면역형광기술검측Nkx2．5、심기기개단백T（ cTnT）적표체。결과단순심기간세포조，생장인자자격후심기간세포수량여형태균무명현변화。여심기공배양조，세포균발생증식여형태변화，Nkx2．5、cTnT양성표체，유개별심기간세포분화위자발박동적심기세포。결론심기간세포여심기세포공배양조건하，HGF화IGF1능구촉진심기간세포증식，연합작용능구유도심기간세포향심기세포분화。
Objective To investigate whether hepatocyte growth factor ( HGF ) and insulin-like growth factor (IGF1) induce cardiac stem cells (CSCs) to proliferate and directly differentiate into cardiomyocytes in vitro.Methods The myocardial tissues were dissected for primary culture of CSCs with the method of explants .The expressions of c-kit and CD34 were examined with immunofluorescence .Primary cells were purified with c-kit by flow cytometry.CFDA SE fluorescent probe was used to detect the proliferation of c-kit+CSCs.C-kit +CSCs were divided into two groups , and cardiac stem cells group and co-cultured with cardiomyocytes group , both group were cultured with HGF and IGF 1.An inverted microscope was used to observe changes in cell number and morphology in different periods .Living cells workstation was used to observe CFDA SE fluorescence intensity , to acquire images and do statistical analysis .Immunofluorescence technique was used to detect the expression of Nkx 2.5 and cardiac troponin T .Results In cardiac stem cells group ,CSCs had no obvious changes in cell number .In co-cultured with cardiomyocytes group , CSCs proliferated and had changes in morphology .Nkx2.5 and cTnT were positively expressed . Several CSCs differentiated into beating cardiomyocytes . Conclusion In co-cultured with cardiomyocytes condition , HGF and IGF1 may promote CSCs to proliferate and differentiate into beating cardiomyocytes .
