叙利亚金黄地鼠血管平滑肌细胞的分离、培养及鉴定

Isolation,culture and identification of vascular smooth muscle cells from Syrian golden hamster

目的分离、培养叙利亚金黄地鼠的血管平滑肌细胞,并对其进行鉴定及生长观察。方法取3~4周龄雄性叙利亚金黄地鼠2只,处死后取主动脉,采用组织块贴壁法对血管平滑肌细胞进行分离、原代和传代培养。采用倒置相差显微镜对原代培养3 d后的细胞及前3代传代细胞进行形态学观察。取第3代对数生长期的血管平滑肌细胞,采用细胞免疫荧光技术观察平滑肌细胞特异性标志因子平滑肌肌动蛋白α(α-SMA)、平滑肌22α(SM22α)染色情况,采用CCK-8法检测细胞培养1~7 d的增殖能力(OD值)。结果原代培养3~5 d,有少量血管平滑肌细胞从组织中爬出,呈贴壁生长;原代培养7~9 d,细胞呈梭形或三角形,生命力旺盛,折光度好;原代培养9 d后,细胞增殖能力明显增强,细胞数量增多,细胞与细胞间排列紧密,出现"峰-谷"状特征;原代培养15 d左右可以进行传代培养。前3代传代细胞与原代细胞相比,形态无明显变化。细胞免疫荧光结果显示,α-SMA、SM22α将细胞质染成绿色,血管平滑肌细胞纯度>95%。血管平滑肌细胞培养1~7 d的OD值分别为0.4373±0.0215、0.4600±0.0188、0.6143±0.0225、0.8453±0.0274、0.8625±0.0240、0.8280±0.0271、0.8160±0.0202。结论成功采用组织块贴壁法分离培养出叙利亚金黄地鼠原代血管平滑肌细胞,细胞传代后2~4 d处于对数生长期,是进行后续实验的最佳时期。

Objective The vascular smooth muscle cells of Syrian golden hamsters were isolated and cultured,and their identification and growth observation were carried out.Methods The vascular smooth muscle cells from two 3-to 4-week-old male Syrian golden hamsters were isolated,primarily cultured and subcultured by tissue attachment method.The cells cultured for 3 days and the cells passed to the third generation were observed by inverted phase contrast microscope.The third generation of vascular smooth muscle cells in the logarithmic phase was taken,and the staining of smooth muscle actin-α(α-SMA)and smooth muscle 22α(SM22α),which were the specific markers of smooth muscle cells,were observed by cellular immunofluorescence technique.The proliferation ability(OD value)of vascular smooth muscle cells cultured for 1-7 days was detected by CCK-8.Results After 3-5 days of primary culture,a small number of vascular smooth muscle cells crawled out of the tissues and grew as adherent;after 7-9 days of primary culture,the cells showed fusiform or triangular shape,exuberant vitality and good refraction;after 9 days of primary culture,the ability of cell proliferation was significantly enhanced,the number of cells increased,and the cells arranged closely with each other,showing the characteristics of"peak-valley",and the primary culture could be subcultured for about 15 days.Compared with the primary cells,there was no significant change in the morphology of the first three passage cells.The results of cellular immunofluorescence showed thatα-SMA and SM22αstained the cytoplasm green,and the purity of vascular smooth muscle cells was more than 95%.The OD values of vascular smooth muscle cells cultured for 1-7 days were 0.4373±0.0215,0.460±0.0188,0.6143±0.0225,0.8453±0.0274,0.8625±0.0240,0.8280±0.0271,and 0.8160±0.0202,respectively.Conclusion The primary vascular smooth muscle cells of Syrian golden hamster are successfully isolated and cultured by tissue attachment method and the the best time for follow-up experiment is 2-4 days after cell passage.