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目的:探讨人大隐静脉(saphenous vein,SV)与胸廓内动脉(internal thoracic artery,ITA)血管平滑肌细胞(VSMCs)的原代培养及传代培养的方法,并比较二者的生长特性。方法:取冠状动脉搭桥术后剩余的SV与ITA血管标本,分为SV组与ITA组,用植块法进行VSMCs的原代培养,用免疫荧光染色法鉴定细胞,并比较两组细胞培养时间的差异。去血清培养48 h后,在DMEM/F12、100 ml/L FBS及10 ng/ml血小板源性生长因子(PDGF)-BB不同培养条件下,观察SV的VSMCs与ITA的VSMCs生长情况。用MTT比色法检测细胞增殖并进行比较。结果:原代及传代培养均获成功。免疫荧光染色法显示平滑肌肌动蛋白(SMα-actin)位于细胞质,总阳性率>95%。SV组原代培养植块周围细胞爬出时间为(9.1±1.1)d,大于ITA组的(5.8±1.0)d(P<0.05)。SV的VSMCs再次传代培养时间为(34.9±3.4)d大于ITA的VSMCs首次传代培养时间(29.1±4.4)d(P<0.05)。SV的VSMCs再次传代培养时间为(9.0±4.2)d,与ITA的VSMCs再次传代培养时间(9.6±3.9)d相似。两组细胞在相同培养条件时,未见明显的形态学差异,细胞生长曲线的差异无统计学意义。结论:采用植块法进行SV与ITA的VSMCs原代培养简便可行,细胞纯度高,具有良好的细胞表型转变特性,是研究冠状动脉搭桥术后血管桥再狭窄分子机制良好的细胞模型。SV的VSMCs可能较ITA的VSMCs具有更强的增殖潜能,二者内在属性的差异仍有待于进一步研究。
OBJECTIVE: To investigate the primary culture and subculturing of saphenous vein (SV) and internal thoracic artery (VSMCs) of human thoracic artery and to compare their growth characteristics. Methods: The remaining SV and ITA blood vessel specimens after coronary artery bypass grafting were divided into SV group and ITA group. Primary culture of VSMCs was performed by explant method. Immunofluorescence staining was used to identify the cells. The cell culture time The difference. After 48 h serum culture, the growth of VSMCs in VSMCs and ITA was observed under different culture conditions of DMEM / F12, 100 ml / L FBS and 10 ng / ml PDGF-BB. Cell proliferation was measured by MTT colorimetric method and compared. Results: Primary and subculture were successful. Immunofluorescence staining showed that SMα-actin was located in the cytoplasm with a total positive rate> 95%. The creeping time of the SV culture group was (9.1 ± 1.1) d, which was greater than that of the ITA group (5.8 ± 1.0) d (P <0.05). VS subcultured in VSMCs for a second time (34.9 ± 3.4) days and subcultured for the first time (29.1 ± 4.4 days) (P <0.05). The VS subculture time of VS was (9.0 ± 4.2) d, which was similar to that of ITA VSMCs subcultured again (9.6 ± 3.9) d. There was no obvious morphological difference between the two groups of cells under the same culture conditions, and the difference of cell growth curve was not statistically significant. CONCLUSION: Primary culture of VSMCs with SV and ITA by the explant method is simple and feasible, with high cell purity and good cell phenotypic transition characteristics. It is a good cell model to study the molecular mechanism of vascular bridge restenosis after coronary artery bypass grafting. VSMCs of SVs may have stronger proliferation potential than VSMCs of ITAs, and the differences in the intrinsic properties of the two remain to be further studied.