BMP9诱导可逆永生化小鼠瓣膜间质细胞成骨能力研究

Osteogenic ability of reversibly immortalized valve mesenchymal cells induced by BMP9 in mice

目的:建立具有快速增殖能力的可逆永生化主动脉瓣膜间质细胞系。方法:体视显微镜下利用弹簧剪夹取瓣膜组织,胶原酶消化后加入逆转录永生化病毒,利用免疫荧光鉴定细胞是否属于间质细胞,显微镜观察细胞形态,结晶紫、CCK-8和流式细胞周期技术检测细胞增殖能力,碱性磷酸酶染色和茜素红钙盐沉积实验检测细胞成骨能力,q-PCR及Western blot检测细胞成骨标志物。结果:经光镜观察和免疫荧光鉴定,成功取到瓣膜间质细胞。加入永生化病毒后,经光镜观察和免疫荧光确认瓣膜间质细胞未发生表型改变,且在永生化的细胞中能够检测到大量SV40T(P<0.001)。结晶紫染色、CCK-8(P<0.001)和流式细胞周期结果提示永生化后的瓣膜间质细胞增殖速度变快。加入成骨诱导因子骨形态发生蛋白-9(bone morphogenetic protein9,BMP9)后永生化瓣膜间质细胞表现出较好的成骨能力,多数成骨标志物在分子层面的RNA和蛋白水平明显升高。去永生化后瓣膜间质细胞形态无明显改变,q-PCR结果显示SV40T明显降低(P<0.001),细胞增殖能力明显下降,且细胞周期被阻滞在G_(0)/G_(1)期。在BMP9诱导下去永生化瓣膜间质细胞同样具有成骨分化能力,且绝大多数成骨标志物都明显上升。结论:本研究成功建立可逆永生化瓣膜间质细胞系,BMP9可诱导其成骨分化,用于后续钙化性主动脉瓣膜疾病研究。

Objective:To establish a reversibly immortalized aortic valve mesenchymal cell line with rapid proliferation.Methods:Valve tissue was clamped with spring scissors under stereomicroscope,and the valve tissue was digested with collagenase and affected with immortalized retrovirus.The cell proliferation ability was detected by crystal violet,CCK-8 and flow cytometry.The osteogenic ability of the cells was detected by ALP staining and alizarin red calcium salt deposition assay.The osteogenic markers were detected by q-PCR and Western blot.Results:By light microscopy and immunofluorescence identification,we confirmed that the mesenchymal cells were successfully obtained.After affected with immortalized virus,light microscopy and immunofluorescence confirmed that there was no phenotypic change in valve mesenchymal cells,and a large amount of SV40T could be detected in immortalized cells(P<0.001).The results of crystal violet staining,CCK-8(P<0.001)and flow cytometry showed that the proliferation rate of valve mesenchymal cells was faster after immortalization.After addition of osteogenic inducible factors BMP9,immortalized valve mesenchymal cells showed better osteogenic ability,and the most of osteogenic markers were significantly increased at the molecular level.There was no significant change in the morphology of valve mesenchymal cells after deimmortalization,and q-PCR results showed that SV40T was significantly reduced(P<0.001).Cell proliferation was significantly decreased,and the cell cycle was arrested in the G_(0)/G_(1)phase.Deimmortalization valve mesenchymal cells induced by BMP9 also had osteogenic differentiation ability,and most osteogenic markers were significantly increased.Conclusion:We have successfully established reversibly immortalized valve mesenchymal cell lines,which can be induced by BMP9 for osteogenic differentiation and can be used for subsequent studies of calcified aortic valve disease.