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目的体外培养、诱导主动脉瓣膜间质细胞钙化,并观察其表型变化。方法使用胶原酶消化法从新鲜猪主动脉瓣膜上分离并体外原代培养主动脉瓣膜间质细胞,行免疫荧光染色细胞鉴定。取4~8代间质细胞,随机分为两组,实验组:以含甘油磷酸、维生素C、地塞米松的钙化培养基进行钙化诱导培养2周;对照组:以标准培养基培养2周。在光学显微镜下行钙化结节计数,茜素红染色观察并半定量检测钙沉积含量;采用实时荧光定量逆转录聚合酶链反应(RT-PCR)检测间质细胞α-平滑肌肌动蛋白(-αSMA)及钙化相关因子(骨钙素、骨桥蛋白、核心结合因子)的基因表达。结果从猪主动脉瓣膜上成功分离并体外培养瓣膜间质细胞,-αSMA及波形蛋白(vimentin)染色阳性,血管性血友病因子(vWF)染色阴性。间质细胞钙化诱导培育2周可成功诱导钙化,自发形成钙化结节,实验组钙化结节(156.25±17.38个/孔vs.2.50±1.29个/孔)和钙沉积(17.52±2.04 vs.1.00±0.22)显著高于对照组(P<0.05);实时RT-PCR提示:实验组-αSMA、骨钙素、骨桥蛋白、核心结合因子等表达均较对照组明显升高(P<0.05)。结论体外诱导钙化的瓣膜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,可能为瓣膜钙化的病理基础。
Objective To culture in vitro and induce the calcification of aortic valve interstitial cells and observe its phenotypic changes. Methods Aortic valves were isolated from fresh porcine aortic valves by collagenase digestion and primary cultured aortic valve interstitial cells in vitro. Immunofluorescent staining was used to identify the cells. Four to eight passages of mesenchymal cells were selected and randomly divided into two groups. Experimental group: calcified medium containing glycerol phosphate, vitamin C and dexamethasone for 2 weeks; control group: cultured for 2 weeks in standard medium . The count of calcified nodules was counted by optical microscope, alizarin red staining was used to detect the contents of calcium deposits, and the content of calcium deposits was detected semi-quantitatively. The expression of α-SMA (-αSMA) in stromal cells was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction ) And calcification related factors (osteocalcin, osteopontin, core binding factor) gene expression. Results The isolated stromal cells were successfully isolated from porcine aortic valves and were positive for α SMA and vimentin staining. VWF staining was negative. The induction of mesenchymal cell calcification induced the calcification within 2 weeks, forming calcified nodules spontaneously. The calcified nodules (156.25 ± 17.38 cells / well vs 2.50 ± 1.29 cells / well) and calcium deposition (17.52 ± 2.04 vs.1.00 ± 0.22) was significantly higher than that of the control group (P <0.05). Real-time RT-PCR indicated that the expressions ofαSMA, osteocalcin, osteopontin and core- . Conclusions The in vitro induced calcification of valve interstitial cells showed a relative activation state, and the phenotypic changes to contractile and osteogenic phenotypes may be the pathological basis of valve calcification.