脐动脉平滑肌细胞体外培养方法的改进

目的:建立并优化的人脐动脉血管平滑肌细胞(VSMCs)的体外培养方法。方法:人脐动脉VSMCs的体外原代培养应用优化的组织块贴壁法进行。在分别应用传统细胞培养法和优化后细胞培养法贴壁培养提取的原代细胞10 d后,计数6孔板每孔细胞数目,并做统计比较分析相同时间内原代细胞的数量。以抗α-平滑肌肌动蛋白(α-SMA)抗体和抗CD31抗体进行免疫荧光染色和倒置相差显微镜观察鉴定培养的细胞。结果:与传统细胞培养法相比,在相同的时间周期内优化实验方法后培养的原代细胞数量较传统多,我们可以在较短时间内得到实验所需的细胞量,因此优化后可缩短细胞的培养周期。原代和传代培养的脐动脉VSMCs生长良好,细胞经冻存、复苏后状态依然良好。光镜下细胞呈典型的"峰-谷"样生长,细胞多为长梭形,具有VSMCs的特征,且传代培养至第7代,细胞生长特性不变。经抗α-SMA、CD31抗体进行免疫荧光染色鉴定证实为VSMCs。结论:优化的组织块贴壁法可缩短原代人脐动脉VSMCs的培养周期,且细胞的生物学特性较稳定,为与平滑肌相关的疾病进展及心血管生长发育的研究奠定了实验基础。

Improvement of in vitro culture method for umbilical artery smooth muscle cells

AIM： To establish and optimize the method for in vitro culture of human umbilical arterial smooth muscle cells （SMCs）. METHODS： We counted each hole cell number in six orifices and statistically analyzed the number of primitive cells within the same time. Human umbilical artery vascular SMCs were cultured in vitro using optimized tissue-pieces inoculation. Alpha smooth muscle actin （alpha- SMA） and CD31 immunofluorescence staining methods and inverted phase contrast microscope were used for cell identification. RESULTS： Compared with the application of traditional cell culture technique, using the optimized cell cultivation this method to extract primitive cells was able to obtain more SMCs at the same time. The primary generation and subculture of SMCs grew well, and after frozen storage and recovery, the typical long spindle ＂peak-valley＂ growth of artery vascular SMCs could still be seen in the cultured cells. With subculture to the seventh generation, cell growth characteristics were stable and the cultured cells were confirmed as SMCs by alpha-SMA and CD31 immunofluorescence staining tests. CONCLUSION： Optimization of the tissue-pieces inoculation shortens the culture cycle time of human umbilical artery SMCs. Cultured cells have stable biological characteristics.