主动脉平滑肌原代细胞提取方法的改良与研究  

Improvement and study on the extraction method of primary aortic smooth muscle cells

作　　者：徐心阳 林文锋 姜烨凡 董志强 李钢 顾嘉玺 倪布清 Xu Xinyang;Lin Wenfeng;Jiang Yefan;Dong Zhiqiang;Li Gang;Gu Jiaxi;Ni Buqing(Department of Cardiovasular Surgery,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院心脏大血管外科,南京210029

出　　处：《中华胸心血管外科杂志》2024年第4期222-227,共6页Chinese Journal of Thoracic and Cardiovascular Surgery

摘　　要：目的改良人主动脉平滑肌细胞的原代提取培养方法。方法采用改良过的组织块贴壁法提取人主动脉平滑肌原代细胞,无多聚左旋赖氨酸(Poly-L-Lysine,PLL)包被为阴性对照组,PLL包被为实验组。采用免疫荧光技术鉴定细胞纯度;蛋白印迹法判断细胞表型变化;细胞计数仪计算传代后细胞的贴壁率;CCK8法鉴定两组细胞生长活力的差别。结果实验组细胞爬出率及成活率明显高于阴性对照组(P<0.05);倒置显微镜及免疫荧光显示两组细胞形态与标志蛋白表达差异无统计学意义;蛋白印迹法显示两组细胞表型改变差异无统计学意义;细胞贴壁率比较,实验组细胞明显高于阴性对照组(P<0.05);CCK8法显示实验组细胞于48、60、72 h时活性明显高于阴性对照组(P<0.05)。结论预包被PLL能够提高人主动脉平滑肌细胞的提取效率、贴壁效率和生长活力,同时不会改变细胞表型。Objective To improve the extraction method for the culture of HASMCs.Methods The primary HASMCs was extracted by modified tissue block adhesion method,and was divided into two groups:the negative control group[without Poly-L-Lysine(PLL)coating]and the experimental group(PLL coating).The cell purity was identified by immunofluorescence technique.The change of cell phenotype was detected by Western Blot.Cell counter was used to calculate the cell adhesion rate after passage.The difference of cell growth vitality between the two groups was distinguished by CCK8 method.Results Creeping out rate and surviving rate of HASMCs in the experimental group were significantly higher than those in the negative control group(P<0.05).Inverted microscope and immunofluorescence showed that there was no remarkable difference in cell morphology and marker protein expression between the two groups.Western blot showed that there was no significant difference in cell phenotype.Compared with the negative control group,the adherent rate of cells in the experimental group was significantly higher(P<0.05).CCK8 method showed that the viability of cells in the experimental group was obviously higher than that in the negative control group at 48 h,60 h and 72 h(P<0.05).Conclusion Pre-coated PLL can improve the extraction efficiency,adhesion efficiency and growth vitality of HASMCs,while it does not change the phenotype of cells.

关 键 词：原代细胞培养 人主动脉平滑肌细胞 多聚左旋赖氨酸 免疫荧光 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]