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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。
Objective: To establish and optimize the culture method of human umbilical artery smooth muscle cells (VSMCs) in vitro. Methods: Human umbilical artery VSMCs primary culture in vitro using optimized tissue block adhesion method. After culturing the extracted primary cells by traditional cell culture method and optimized cell culture method respectively for 10 days, the number of cells per well in the 6-well plate was counted and the number of primary cells in the same time was statistically analyzed. Cultured cells were identified by immunofluorescence staining and inverted phase contrast microscopy with anti-α-smooth muscle actin (α-SMA) antibodies and anti-CD31 antibodies. Results: Compared with the traditional cell culture method, the number of primary cells cultured after optimization of the experimental method in the same time period is more than that of the traditional method. Therefore, we can obtain the required amount of cells in a short period of time, The incubation period. Primary and subculture cultured umbilical artery VSMCs grew well, the cells were frozen, and the recovery was still good. Under light microscope, the cells showed the typical “peak-valley” growth. Most of the cells were long fusiform and had the characteristics of VSMCs. The cells were subcultured to the seventh passage, and the cell growth characteristics were unchanged. The anti-α-SMA, CD31 antibody immunofluorescence staining identified as VSMCs. CONCLUSION: Optimized tissue block adhesion method can shorten the culture period of primary human umbilical artery VSMCs, and the biological characteristics of the cells are stable, which lays an experimental foundation for the study of smooth muscle related diseases and cardiovascular growth and development.