主动脉瓣体外钙化模型的建立

An induced calcification model of aortic valvular cells in vitro

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 construct an induced calcification model of aortic valves by isolation and inducting calcification in porcine aortic valvular interstitial cells in vitro.Method：Porcine aortic valvular interstitial cells（VIC） were isolated and expanded by collagenase methods,and identified by immunofluorescence staining.VICs at 4-8 passages were cultured in osteogenic media to induce calcification for up to 2weeks.Calcified nodules were quantified.Calcium deposit was stained and measured by Alizarin Red S staining and assay.Real time reverse transcription polymerase chain reaction（Real Time rt-PCR）was performed to measure expression of alpha smooth muscle actin（α-SMA）and calcification related factors（osteocalcin,osteopontin and cbfa1）.Result：VICs were successfully harvested from porcine aortic valves,indentified by positive staining ofα-SMA,vimentin and negative staining of von Willebrand factor.VICs could calcify after 2weeks of osteogenic induction with calcified nodules formed that positively stained by Alizarin Red S.Quantification of nodules and calcification deposit was higher in experimental group than in control group.Real Time rt-PCR indicated that calcification was associated with upregulation ofα-SMA expression,as well as calcification related markers like osteocalcin,osteopontin and Cbfa1/Runx-2.Conclusion：The in vitro aortic calcification model could be induced and constructed by use of collagenase methods and osteogenic media.