血管外膜细胞钙化及其钙化机制研究  

Calcification of Vascular Adventitial Cells and Its Mechanism

作　　者：谭小青 张旭升[1] 樊小容 黄战军[1] TAN Xiaoqing;ZHANG Xusheng;FAN Xiaorong;HUANG Zhanjun(Longgang District People′s Hospital of Shenzhen,Shenzhen 518172,Guangdong,China)

机构地区：[1]深圳市龙岗区人民医院,广东深圳518172

出　　处：《中西医结合心脑血管病杂志》2023年第18期3347-3350,共4页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

摘　　要：目的:研究经体外诱导钙化建立大鼠血管外膜细胞钙化模型,检测钙化过程中成骨相关指标及凋亡、自噬相关蛋白的表达变化,旨在为心血管疾病模型提供更精确的细胞模型,并初步探讨其钙化机制。方法:原代提取大鼠胸主动脉外膜纤维细胞,取3~6代细胞使用诱导培养基(高糖DMEM+10%胎牛血清+10 mmol/Lβ-甘油磷酸+0.05 mmol/L抗坏血酸+100 mmol/L地塞米松)诱导钙化,诱导时间为3 d、6 d、9 d、12 d、15 d,筛选出诱导细胞钙化的最佳时间。对细胞采用茜素红S染色、细胞内钙含量测定和碱性磷酸酶(ALP)活性检测,鉴定是否成功构建钙化模型。采用实时定量聚合酶链式反应(PT-PCR)检测成骨相关因子骨形态生成蛋白2(BMP2)和核心结合因子α1(Runx2)的mRNA含量,蛋白免疫印迹法(Western Blot)检测凋亡蛋白Bax、Bcl-2和自噬相关蛋白微血管相关蛋白(LC3)、Beclin-1的表达水平,找出血管外膜细胞钙化的潜在机制。结果:当诱导钙化时间为15 d时,血管外膜细胞中主要钙化指标胞内钙含量及ALP活性上调(P<0.05),茜素红S染色显示钙化组有明显钙盐沉积。血管外膜细胞经钙化诱导后,BMP2和Runx2的mRNA水平上调,Bax蛋白水平上调,Bcl-2和Beclin-1蛋白水平下调,LC3-Ⅱ/LC3-Ⅰ比值上调(P<0.05)。结论:钙化诱导培养基培养血管外膜细胞15 d可成功构建钙化细胞模型,血管外膜细胞钙化可能与细胞向成骨样表型转化有关,血管外膜细胞钙化过程涉及细胞自噬及凋亡调控。Objective:To investigate the mananism of calcification of rat vascular adventitial cells,establish the calcification model of rat vascular adventitial cells,and detect the expression changes of osteogenesis-related indicators,apoptosis,and autophagy-related proteins during the calcification process.It aimed to provide more accurate cell models for cardiovascular disease and initially explore the mechanism of calcification.Methods:Rat thoracic aortic adventitial fibroblasts were extracted from the primary generation,and the 3rd to 6th generation cells were used for induction medium(high glucose DMEM+10%fetal bovine serum+10 mmol/Lβ-glycerophosphate+0.05 mmol/L ascorbic acid+100 mmol/L dexamethasone)to induce calcification,the induction time was 3,6,9,12,and 15 d,and the optimal time for inducing cell calcification was selected.The cells were stained with alizarin red S,detected by intracellular calcium content and alkaline phosphatase(ALP)to identify whether the calcification model was successfully constructed.Real-time quantitative reverse transcription polymerase chain reaction(RT-PCR)was used to detect the mRNA levels of osteogenesis-related factors bone morphogenetic protein(BMP2)and runt-related transcription factor 2(Runx2);Western Blot was used to detect the apoptosis proteins Bax,Bcl-2,the autophagy-related proteins LC-3,and Beclin-1 expression level;then the potential mechanism of vascular adventitial cell calcification would be revealed.Results:When calcification was induced for 15 days,the intracellular calcium content in the adventitial cells of the main calcification indicators and ALP activity were up-regulated(P<0.05).Alizarin red S staining showed obvious calcium deposits in the calcification group.After calcification was induced in adventitial cells,the mRNA levels of BMP2 and Runx2 up-regulated,the protein levels of Bax up-regulated,the protein levels of Bcl-2 and Beclin-1 down-regulated,and the ratio of LC3-Ⅱ/LC3-Ⅰdown-regulated(P<0.05).Conclusion:Adventitial cells cultured in the calcifica

关 键 词：血管外膜细胞 钙化 成骨样表型转化 自噬与凋亡 实验研究 

分 类 号：R543[医药卫生—心血管疾病]