摘要
目的探讨体外培养胎鼠心肌细胞方法的优化。方法取孕19日SD胎鼠分离心肌细胞进行原代培养,采用0.8 g/L胰蛋白酶和0.4 g/L 2型胶原蛋白酶联合消化后,以多次差速分离,不同种类的动物血清(胎牛血清组、马血清组)改良心肌细胞培养条件。倒置相差显微镜下观察心肌细胞形态及搏动频率、免疫荧光细胞化学染色分别检测α-横纹肌辅肌动蛋白(α-SA)与心肌肌钙蛋白Ⅰ(cTnⅠ)鉴定胎鼠心肌细胞及培养24、48、72、96 h的心肌细胞纯度。结果心肌细胞接种24 h,可见大部分贴壁细胞和少量的悬浮细胞,亦见少许搏动的心肌细胞;48、72、96 h,马血清组心肌细胞α-肌动蛋白及cTnⅠ阳性率明显高于胎牛血清组。结论采用胰蛋白酶和2型胶原蛋白酶联合消化和马血清更易培养出胎鼠心肌细胞。
Objective To explore the optimal method for in vitro cultivation of SD fetal rat cardiomyocytes. Methods Cardiomyocytes of SD fetal rats from pregnant rats on gestational day 19 were obtained by digesting cardiac tissues with 0. 8 g/L trypsin and 0. 4 g/L collagenase Ⅱ. With differential centrifugation and different animal serums（ bovine fetal serum or horse serum）,we tried to look for the optimal culture conditions. The morphology and beat frequency of cardiomyocytes were observed under an inverted phase-contrast microscope. In addition, the expressions of alpha-sarcomeric actinin（α-SA） and cardiac troponin I（cTnⅠ） in the cultured cardiomyocytes were detected by immunofluorescence staining to identify cardiomyocytes and the purity of these cells after 24,48,72 and 96 hours of cultivation. Results After 24 hours of cultivation,we had seen a majority of adherent cells and a few of suspension cells,and also seen some pulsating cardiomyocytes. The positive rates of α-SA and cTnⅠ in the cardiomyocytes of the horse serum-cultured group were obviously higher than those in the bovine fetal serum-cultured group after 48,72 and 96 hours of cultivation. Conclusion Co-digestion with trypsin and collagenaseⅡ in combination with horse serum cultivation are easier to cultivate fetal rat cardiomyocytes.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第11期1534-1538,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
广州医科大学博士启动/留学归国人员科研项目(2013C59)
广东省科技计划项目(20140212)
广州市医药卫生科技项目(20151A011094
20161 A011091)
