摘要
目的研究血管平滑肌细胞钙化中受体相互作用蛋白激酶3(Ripk3)调控钙磷沉积、细胞骨分化和细胞凋亡的作用。方法采用β磷酸甘油和氯化钙培养大鼠主动脉血管平滑肌细胞诱导钙化。实验随机分为4组:对照组、钙化组、Ripk3^(-/-)组、钙化+Ripk3^(-/-)组(联合组)。使用茜素红S染色检测血管平滑肌细胞钙化情况,同时测定细胞内Ca^(2+)含量和碱性磷酸酶活性。Western blot法测定Runt相关转录因子2、骨形态发生蛋白2和半胱氨酸天冬氨酸蛋白酶3表达。结果血管平滑肌细胞钙化3、7 d的Ripk3表达较钙化前明显增加,14 d的Ripk3表达较7 d明显增加,并达到最高值,差异有统计学意义(P<0.05)。与对照组和Ripk3^(-/-)组比较,钙化组Ca^(2+)、碱性磷酸酶活性、Runt相关转录因子2、骨形态发生蛋白2、细胞凋亡率和活化的半胱氨酸天冬氨酸蛋白酶3表达明显增加,差异有统计学意义(P<0.05)。与钙化组比较,联合组Ca^(2+)和碱性磷酸酶活性明显降低[(102.8±12.8)nmol/mg vs(457.1±51.2)nmol/mg,(136.1±15.4)U/mg vs(412.2±46.7)U/mg,P<0.05],Runt相关转录因子2和骨形态发生蛋白2表达明显降低(1.07±0.15 vs 1.84±0.23,1.27±0.14 vs 3.01±0.25,P<0.05)。与钙化组比较,联合组细胞凋亡率和活化的半胱氨酸天冬氨酸蛋白酶3表达明显降低,差异有统计学意义[(15.8±3.9)%vs(31.1±4.2)%,1.19±0.14 vs 2.21±0.23,P<0.05]。结论Ripk3通过促进钙磷沉积、细胞骨分化和细胞凋亡起到加重血管钙化的作用。
Objective To study the role of receptor interacting protein kinase 3(Ripk3)in regulating deposition of calcium and phosphorus,differentiation and apoptosis of vascular smooth muscle cells(VSMC).Methods The VSMC were cultured inβ-glycerophosphate and calcium chloride to induce their calcification.The VSMC were divided into control group,calcification group,Ripk3 knockout group and calcification+Ripk3 knockout group.The calcification of VSMC was detected with Alizarin red staining.The intracellular Ca^(2+)level and alkaline phosphatase activity were measured by ELISA.The expressions of Runt-related transcription factor 2,bone morphogenetic protein 2 and Caspase-3 were detected by Western blot.Results The expression level of Ripk3 was significantly higher on days 3 and 7 after the calcification of VSMC than before the calcification of VSMC(P<0.05)and on day 14 than on day 7 after the calcification of VSMC and reached its peak on day 14 after the calcification of VSMC(P<0.05).The activity of Ca^(2+)and alkaline phosphatase,expression levels of Runt-related transcription factor 2,bone morphogenetic protein 2,activated Caspase-3,and apoptosis rate of VSMC were significantly higher in calcification group than in control group and Ripk3 knockout group(P<0.05).The activity of Ca^(2+)and alkaline phosphatase and the expression levels of Runt-related transcription factor 2 and bone morphogenetic protein 2 were significantly lower in calcification+Ripk3 knockout group than in calcification group(102.8±12.8 nmol/mg vs 457.1±51.2 nmol/mg,136.1±15.4 U/mg vs 412.2±46.7 U/mg,P<0.05;1.07±0.15 vs 1.84±0.23,1.27±0.14 vs 3.01±0.25,P<0.05).The apoptosis rate of VSMC and expression levels of Caspase-3 were significantly lower in calcification+Ripk3 knockout group than in calcification group(15.8%±3.9%vs 31.1%±4.2%,1.19±0.14 vs 2.21±0.23,P<0.05).Conclusion Ripk3 aggravates the calcification of VSMC by promoting the deposition of calcium and phosphorus,the apoptosis and differentiation of VSMC.
作者
陈韦任
吴雪萍
周玉杰
钱赓
沙媛
王锦达
Chen Weiren;Wu Xueping;Zhou Yujie;Qian Geng;Sha Yuan;Wang Jinda(Department of Cardiology,Affiliated Beijing Anzhen Hospital of Capital Medical University,Beijing Institute of Heart,Lung and Blood Vessel Disease,Beijing 100029,China)
出处
《中华老年心脑血管病杂志》
北大核心
2021年第5期523-526,共4页
Chinese Journal of Geriatric Heart,Brain and Vessel Diseases
基金
国家重点研发计划项目(2017YFC0908800)
北京市医院管理局“使命”计划专项经费资助(SML20180601)
北京市博士后资助项目(202011)。
