兔血管平滑肌细胞体外培养及生长特性研究

目的体外分离培养兔血管平滑肌细胞（VSMC）并观察其生长特征。方法采用酶消化结合贴壁法原代培养兔胸主动脉来源的VSMC并传代,倒置显微镜观察和免疫荧光染色法鉴定培养细胞;锥蓝法、绘制生长曲线、MTT法和划痕法分别测定VSMC传代细胞的成活率及其生长、增殖和迁移能力。结果原代培养5d后,VSMC从组织块边缘长出,传代细胞呈典型＂峰-谷＂状生长,胞质内α-平滑肌肌动蛋白免疫荧光染色阳性;细胞成活率为96%;生长曲线近似＂S＂形;细胞生长第3～5d内光密度值变化较明显;无血清培养的VSMC在24h内划痕宽度变化最显著。结论体外培养的兔动脉平滑肌细胞为收缩表型且活性高,生长3～5d细胞增殖活力较强,无血清培养的细胞在24h内迁移能力最强,为心血管疾病研究提供了良好的实验材料。

In vitro culture and growth characteristics of rabbit vascular smooth muscle cells

Objective To observe in vitro culture and grow characteristics of rabbit artery  smooth muscle cells. Methods Digestive and adherent method was applied for the primary culture of vascular smooth muscle cells (VSMC) deriving from rabbit thoracic aorta, and cells were passaged. The cultured cells were observed and identified by inverted microscopy and immunofluorescence method. Viability, growth ability, proliferation ability and migration ability of passage cells were determined by trypan blue exclusion method, growth curve, MTT assay and wound healing assay. Results VSMC of primary culture grew out of the edge of tissue pieces after incubation for 5 d. The passage cells maintained typical “hill” and “valley” pattern of growth observed by light microscope. Positive staining of α-actin in cell cytoplasm was revealed by immunofluorescence. Cell viability was 96%, the growth curve of VSMC resembled “S” in shape, the significant change of optical density was detected after culture for 3 to 5 d, and the scarification width value changed markedly in VSMC treated by serum starvation for 24 h. Conclusion The cultured rabbit artery smooth muscle cells are characterised by contractile phenotype and better viability, with higher proliferation activity after growth for 3 to 5 d and migration ability within 24 h after scarification, which may provide favourable experimental material for the research of cardiovascular diseases.