CC趋化因子受体2在瓣膜间质细胞成骨转化中的作用及其机制

Mechanism of CC chemokine receptor 2 in osteogenic transformation of valvular mesenchymal cells

目的探讨CC趋化因子受体2(CCR2)在瓣膜间质细胞成骨转化中的作用及其机制。方法2020年6月东南大学附属中大医院实验室通过胶原酶消化法改良后实施体外分离大鼠瓣膜间质细胞后进行培养、鉴定,并以随机抽签法将细胞分成试验组及对照组。对照组细胞培养基中加入2 ml的RPMI 1640完全培养液,试验组细胞培养基中则加入RPMI 1640完全培养液以及1.0 ng/ml的MCP-1。比较两组细胞中CCR2蛋白表达水平,碱性磷酸酶(ALP)、骨形态发生蛋白(BMP)-2蛋白与内参灰度比值,ALP、BMP-2基因与内参灰度比值,采用Pearson法进行相关性的分析。结果试验组细胞中CCR2蛋白表达水平高于对照组(1.549±0.392比0.742±0.257,t=5.444,P<0.05)。试验组ALP蛋白与内参灰度比值明显高于对照组(0.732±0.052比0.189±0.029,t=28.840,P<0.05)。试验组BMP-2蛋白与内参灰度比值明显高于对照组(0.891±0.058比0.401±0.033,t=23.220,P<0.05)。试验组ALP基因与内参灰度比值明显高于对照组(0.248±0.006比0.104±0.003,t=67.882,P<0.05)。试验组BMP-2基因与内参灰度比值明显高于对照组(0.518±0.048比0.198±0.039,t=16.362,P<0.05)。CCR2蛋白表达量与ALP蛋白和内参灰度比值(r=0.513,P<0.05)、BMP-2蛋白和内参灰度比值(r=0.513,P<0.05)、ALP基因和内参灰度比值(r=0.602,P<0.05)、BMP-2基因和内参灰度比值(r=0.616,P<0.05)均呈正相关关系。结论CCR2可促进瓣膜间质细胞向成骨样细胞转化。

Objective To analyze the mechanism of CC chemokine receptor 2(CCR2)in osteogenic transformation of valvular mesenchymal cells.Methods In June,2020,after the improvement of collagenase digestion,the interstitial cells of rat valve were isolated in vitro,cultured and identified,and the cells were divided into experimental group and control group by random lottery.2 ml RPMI 1640 complete culture medium was added to the control group cell culture medium,while RPMI 1640 complete culture medium and 1.0 ng/ml MCP-1 were added to the experimental group cell culture medium.The expression level of CCR2 protein,the gray ratio of alkaline phosphatase(ALP),bone morphogenetic protein-2(BMP-2)protein and internal reference,and the gray ratio of ALP,BMP-2 gene and internal reference were compared between the two groups.Pearson method was used to analyze the correlation.Results The expression level of CCR2 protein in experimental group was higher than that in control group(1.549±0.392 vs.0.742±0.257,t=5.444,P<0.05).The gray ratio of ALP protein to internal reference in experimental group was significantly higher than that in control group(0.732±0.052 vs.0.189±0.029,t=28.840,P<0.05).Gray scale ratio of BMP-2 protein to internal reference in experimental group was significantly higher than that in control group(0.891±0.058 vs.0.401±0.033,t=23.220,P<0.01).The gray ratio of ALP gene to internal reference in experimental group was significantly higher than that in control group(0.248±0.006 vs.0.104±0.003,t=67.882,P<0.05).Gray scale ratio of BMP-2 gene to internal reference in experimental group was significantly higher than that in control group(0.518±0.048 vs.0.198±0.039,t=16.362,P<0.05).The ratio of CCR2 protein expression to ALP protein and internal reference gray scale(r=0.513,P<0.05),BMP-2 protein and internal reference gray scale(r=0.513,P<0.05),ALP gene and internal reference gray scale(r=0.602,P<0.05),BMP-2 gene.Conclusion CCR2 can promote the transformation of valvular mesenchymal cells into osteoblasts,which may be one of the new targets for the prevention and treatment of aortic valve calcification.