摘要
目的:建立新生SD大鼠c-kit阳性细胞分离纯化及传代的培养方法。方法:取出生24h之内新生SD大鼠心尖部组织,用PBS液洗去心尖部的血液,将其剪碎成lmm3以下的组织块培养,培养48h后,0.5%Trypsin-EDTA消化,用免疫磁珠分选c-kit阳性的心肌细胞,继续培养传代。用倒置显微镜观察c-kit阳性心肌细胞,用免疫细胞化学法检测心肌组织特异性cTnT及Desmin蛋白。结果:用心脏组织培养法成功得到了体外搏动的原代心肌细胞;用免疫磁珠分选了c-kit阳性心肌细胞,倒置显微镜下观察到c-kit阳性心肌细胞具有传代增殖的特性;同时,检测了c-kit阳性心肌细胞胞质cTnT及Desmin结合蛋白的阳性表达。结论:采用改良后的培养方法获得了c-kit阳性心肌细胞,该细胞具有心肌干细胞的蛋白表达特性,并具有一定的传代增殖能力。
objective To establish a method about separation, purification and passage of c - kit ＋ myocardial cells in SD newborn rats. Method Cardiac apex that removed from newborn SD rat within 24 hours washed by PBS, and cut it about lmm3, cultivate after 48 hours, 0. 5% Trypsin- EDTA digestion, c- kit＋ myocardial cells separated by positive immune magnetic bead , continuing sub- culture. To observe the c - kit＋ myocardial cell growth, and immunocytochemistry detection of cTnT and Desmin protein. Results Pulsatile primary cardiomyocytes in vitro have been successfully gotten with cardiac tissue culture method; c - kit ＋ myocardial cells have separated by positive immune magnetic bead, The passaged proliferation characteristics of c - kit - positive cardiac cells have been detected in an inverted microscope. Meanwhile, the positive expression of cTnT and Desmin in the cytoplasmic of c - kit - positive cardiac cells have been detected. Conclusion C - kit＋ myocardial cells, with the protein expression characteristics of myocardial stem cell and the proliferation ability , have been obtained by the cultivation of the improved method.
出处
《中国民族民间医药》
2014年第18期15-16,18,共3页
Chinese Journal of Ethnomedicine and Ethnopharmacy
基金
重庆教委科学技术研究项目(KJ101804)
