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目的:探讨一种体外分离、扩增瓣膜间质细胞并诱导钙化的方法,建立体外瓣膜细胞钙化模型。方法:采用胶原酶消化法从新鲜猪主动脉瓣膜上分离并体外扩增瓣膜间质细胞,免疫荧光染色行细胞鉴定。4～8代间质细胞以含β-甘油磷酸的钙化培养基钙化诱导培养2周。钙化结节计数,茜素红S染色观察并检测钙沉积。实时定量逆转录聚合酶链反应(Real Time rt-PCR)检测α-平滑肌波动蛋白(α-SMA)及钙化相关因子骨钙素、骨桥蛋白、核心结合因子a1(Cbfa1)表达。结果:胶原酶消化法可从猪主动脉瓣膜上成功分离并体外扩增瓣膜间质细胞,α-SMA和波形蛋白(Vimentin)免疫荧光染色阳性,血管性血友病因子(vWF)染色阴性。钙化培养基体外钙化诱导培养1～2周可成功诱导钙化,间质细胞自发形成钙化结节,茜素红S染色阳性,钙沉积明显增加(P<0.05)。实时定量逆转录PCR提示钙化间质细胞α-SMA表达上调,并相对高表达钙化相关因子(P<0.05)。结论:胶原酶法联合钙化培养基可成功体外构建主动脉瓣膜细胞钙化模型。
OBJECTIVE: To explore a method for isolating and amplifying valve interstitial cells and inducing calcification in vitro, and to establish an in vitro model of valve cell calcification. Methods: Collagenase digestion method was used to separate and purify valvular interstitial cells from fresh porcine aortic valves. Immunofluorescence staining was used to identify the cells. Four to eight passages of the mesenchymal cells were cultured for two weeks under calcification with β-glycerophosphate-containing calcification medium. Calcified nodules were counted, Alizarin Red S staining was observed and calcium deposition was measured. The expressions of α-smooth muscle actin (α-SMA) and calcitonin-related osteocalcin, osteopontin and corebinding factor a1 (Cbfa1) were detected by real-time RT-PCR. RESULTS: Collagenase digestion successfully isolated and amplified extracorporeal interstitial cells from porcine aortic valves. Α-SMA and Vimentin immunofluorescence staining showed that vWF staining was negative. In Vitro Calcification of Calcified Medium Calcification was induced in 1 ~ 2 weeks after induction of culture. Mesenchymal cells formed calcified nodules spontaneously. Alizarin red S staining was positive and calcium deposition was significantly increased (P <0.05). Real-time quantitative reverse transcription PCR showed that the expression of α-SMA in calcified stromal cells was up-regulated and the calcification-related factors were relatively high (P <0.05). Conclusion: Collagenase combined with calcification medium can successfully construct aortic valve calcification model in vitro.