Notch2高表达可使人主动脉平滑肌细胞骨形态发生蛋白4表达增加促进钙化

Over-expression of Notch2 Increases BMP-4 Expression and Promotes Calcification in Human Aortic Smooth Muscle Cells

目的观察并验证人主动脉平滑肌细胞(HASMC)Notch2蛋白表达的变化对其细胞钙化的影响,并探讨其机制。方法体外培养HASMC,将其分为两组,分别为正常对照组与诱导钙化组。前者常规培养,后者在常规培养的基础上加用钙化诱导剂β-甘油磷酸(5 mmol/L)、抗坏血酸(50μg/mL)和地塞米松(100 nmol/L),两组细胞均培养7 d。另取上述方法诱导钙化的HASMC为钙化对照组,再设钙化抑制组,即在钙化对照组的基础上加入Notch特异性抑制剂γ-内分泌酶抑制剂(50μmol/L),两组细胞均培养7 d。四组均用白介素-6诱导细胞的炎症反应。采用免疫印迹法检测各组细胞内Notch2、磷酸化核转录因子STAT3(p-STAT3)和骨形态发生蛋白4(BMP-4)的蛋白表达水平;酶联免疫法测定各组细胞培养上清液中BMP-4蛋白的含量;逆转录-聚合酶链反应检测诱导钙化组和正常对照组中HASMC Notch2 mRNA的表达;免疫荧光染色法观察钙化对照组与钙化抑制组中HASMC BMP-4蛋白的表达。结果(1)诱导钙化组中HASMC Notch2、p-STAT3和BMP-4蛋白表达水平明显高于正常对照组(P<0.05);诱导钙化组的培养液上清中BMP-4蛋白含量为(66.42±2.89)pg/mL,明显高于正常对照组的(22.35±2.68)pg/mL(P<0.05);诱导钙化组中HASMC Notch2 mRNA的表达明显高于正常对照组(P<0.05)。(2)钙化抑制组中HASMC Notch2、p-STAT3和BMP-4蛋白的表达水平明显低于钙化对照组(P<0.05);钙化抑制组培养液上清中BMP-4蛋白的含量为(22.34±3.82)pg/mL,明显低于钙化对照组的(62.89±3.26)pg/mL(P<0.05)。免疫荧光染色显示钙化抑制组中HASMC BMP-4蛋白表达明显低于钙化对照组。结论钙化的HASMC Notch2蛋白表达增高,促进了BMP-4蛋白表达增加,从而使HASMC发生成骨样改变,使血管发生钙化,这一机制可能与JAK-STAT3的活化有关。

Objective To observe the effect of Notch2 protein expression on calcification of human aortic smooth muscle cells(HASMC)and explore its mechanism.Methods HASMC was cultured in vitro and divided into two groups:normal control group and induced calcification group.The former were cultured in conventional media.The latter was supplemented with calcification inducers β-glycerophosphate(5 mmol/L),ascorbic acid(50μg/mL)and dexamethasone(100 nmol/L)on the basis of conventional culture.The cells of two group were cultured for 7 days.HASMC,which was induced calcification by the above-mentioned method,was taken as the calcification control group,and the calcification inhibition group was set up,in which Notch specific inhibitor-endocrine enzyme inhibitor(50μmol/L)was added to the calcification control group.Interleukin-6 was used to induce the inflammatory reaction of the cells in all four groups.The expression levels of Notch2,phosphorylated nuclear transcription factor STAT3(p-STAT3)and bone morphogenetic protein-4(BMP-4)were detected by Western blotting and BMP-4 protein was detected by ELISA in all groups.The expression of Notch2 mRNA was detected by RTPCR in induced calcification group and control group,and the expression of HASMC BMP-4 protein was detected by immunofluorescence staining in calcification inhibition group and calcified control group.Results(1)The expression of Notch2,p-STAT3 and BMP-4 in induced calcification group is significantly higher than that of the control group(P<0.05).The expression of BMP-4 in the induced calcification group is(66.42±2.89)pg/mL,which is significantly higher than that in control group[(22.35±2.68)pg/mL](P<0.05).Notch2 mRNA expression in induced calcification group is significantly higher than that in normal control group(P<0.05).(2)The expression of Notch2,p-STAT3 and BMP4 protein in the calcification inhibition group was significantly lower than that in the calcification control group(P<0.05).The content of BMP-4 in supernatant of calcification inhibition group was(22.34±3.82)pg/mL,which was significantly lower than that of calcification control group[(62.89±3.26)pg/mL](P<0.05).Immunofluorescence staining showed that BMP-4 protein expression was significantly lower in calcification inhibition group than that in calcified control group.Conclusion The increased expression of calcified HASMC Notch2 promotes the expression of BMP-4,which leads to osteogenic changes of HASMC and calcification of blood vessels.This mechanism may be related to the activation of JAK-STAT3.