人主动脉瓣间质细胞原代培养及体外钙化模型的建立  

Primary culture and in vitro calcification model establishment of human aortic valve interstitial cells

作　　者：张米[1] 刘晓红[1] 张伯尧[1] 韩林[1] 陆方林[1] 张锡武[1] 龚德军[1] 徐志云[1] 

机构地区：[1]第二军医大学长海医院胸心外科,上海200433

出　　处：《第二军医大学学报》2013年第5期488-492,共5页Academic Journal of Second Military Medical University

基　　金：上海市基础研究重点项目(11JC1415900)~~

摘　　要：目的原代培养人主动脉瓣间质细胞并建立体外瓣膜细胞钙化模型,诱导人主动脉瓣间质细胞向成骨细胞分化,并观察其表型变化。方法采用胶原酶两次消化法原代培养人主动脉瓣间质细胞,取传代3～7代间质细胞,随机分为2组,实验组以钙化诱导培养基培养,对照组以标准培养基培养。1周后,行von Kossa染色观察钙化结节形成情况,分光光度计测定碱性磷酸酶活性,免疫荧光染色检测瓣膜间质细胞表型蛋白,real-time PCR及蛋白质印迹分析检测成骨相关因子的表达,评价模型建立情况。结果培养1周后实验组出现钙化结节,每孔钙化结节数量[(51.20±14.31)个]高于对照组[(3.60±1.82)个],差异有统计学意义(P<0.05),同时实验组碱性磷酸酶活性较对照组升高(约上升4倍,P<0.05),细胞收缩表型平滑肌肌动蛋白(α-SMA)增高。Real-time PCR及蛋白质印迹分析提示,实验组中成骨相关因子Runx2、osteocalcin及osteopontin在mRNA及蛋白水平均较对照组升高,磷酸化Smad1/5/8蛋白表达也同时升高,差异有统计学意义(P<0.05)。结论成功建立了人主动脉瓣间质细胞体外诱导钙化模型,诱导后间质细胞呈现相对激活状态,表型向收缩表型和成骨表型转化,为今后实验提供了可靠的细胞模型。Objective To primary culture human aortic valvular interstitial cells （hVICs）, establish their in vitro calcification model, and to induce hVICs differentiation to osteogenesis and to observe the phenotype changes. Methods hVICs were digested from native valves and used for experiments after 3-7 passages. The cells were cultured in osteogenic media or in normal media. One week later the calcified nodules were stained and measured by yon Kossa. The activity of alkaline phospbatase （ALP） was examined by spectrophotometer, immunofluorescence staining was used to detect the phenotype protein of hVfCs, and real-time PCR and Western blotting analysis were used to examine the osteogenesis associated factors to assess the calcification model of hVICs. Results The calcified nodules were found 7 days after osteogenic induction. The calcified nodules in the experimental group were significantly more than that in the control group （51.20±14.31/well vs 3.60±1.82/ well,P〈0.05）. The activity of ALP was significantly increased after osteogenic induction compared with the control group （increased by about 4 folds, P〈0.05）, with increased contractile phenotype a-smooth muscle actin （α-SMA）. Real time PCR and Western blotting results indicated that the expressions of Runx2, osteocalcin, and osteopontin in the experiment group were significantly higher than those in the control group （P〈0. 05）, so was the expression of phosphorylated Smadl/5/8（P〈0. 05）. Conclusion We have successfully established the in vitro calcification model of hV1Cs, with hVICs in an activated state; and the phenotype shifts to contraction and ossification, which provide a reliable cell model for the future study.

关 键 词：主动脉瓣 间质细胞 细胞培养技术 细胞表型 

分 类 号：R329.29[医药卫生—人体解剖和组织胚胎学]