目的: 建立一种简单、高效的小鼠主动脉血管平滑肌细胞(vascular smooth muscle cells，VSMCs)原代培养方法及体外钙化模型。方法: 采用改良的组织贴块法培养小鼠VSMCs，经免疫荧光染色法鉴定后，将细胞随机分为对照组和钙化组。采用von kossa染色及比色法检测两组细胞内钙含量。结果： 培养3~5 d时，可见细胞从组织块边缘爬出，7~10 d后细胞融合成片；免疫荧光染色显示胞质表达特异性α-肌动蛋白；VSMCs培养至第2代细胞纯度达95%；与对照组相比，钙化组细胞的钙含量、碱性磷酸酶（ALP）活性明显增加(P均<0.05)。结论： 改良的组织贴块法可获得高纯度的小鼠VSMCs；β-甘油磷酸钠在体外可有效诱导其钙化。
Abstract
Objective: To establish a simple and efficient method for primary culture of mouse aortic vascular smooth muscle cells (VSMCs) and calcification model in vitro. Methods: The primary VSMCs were harvested by modified tissue-piece inoculation and identified by immunocytochemistry. The established VSMCs were randomly divided into control group and calcification group. Calcification of cells was assayed by von kossa staining and colorimetry method. Results: VSMCs migrated from explants of mouse aorta tissue after 3-5 days of culture. After 7-10 days, VSMCs grew to form a fusing monolayer.Immunofluorescence staining with specific mAb against mouse α-actin demonstrated VSMCs were positive. The cell purity of the 2nd generation of VSMCs was over 95%. Compared with the control group, the calcium content and ALP activity in the calcification group were increased significantly (both P<0.05). Conclusion: The method of modified explant-culture successfully established a effective model for primary culture of VSMCs, of which calcification in vitro could be induced by β-glycerophosphate.
关键词
血管平滑肌细胞 /
原代培养 /
钙化
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参考文献
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脚注
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基金
国家自然科学基金资助项目(81170279，81370409)；江苏省自然基金及创新团队基金资助项目(BK2011486，LJ201116)；镇江市心血管病重点实验室(SS2012002)
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