斑蝥素对血小板衍生生长因子BB诱导血管平滑肌细胞增殖和迁移的机制研究  

Mechanism of cantharidin underlying proliferation and migration of vascular smooth muscle cells induced by PDGF-BB

作　　者：邱立强 徐昌武[1] 李雯静 江洪[1] 吴刚[1] 罗强 夏豪[1] Qiu Liqiang;Xu Changwu;Li Wenjing;Jiang Hong;Wu Gang;Luo Qiang;Xia Hao(Department of Cardiology,Wuhan University People's Hospital,Wuhan 430060,Hubei Province,China)

机构地区：[1]武汉大学人民医院心血管内科,430060 [2]武汉科技大学附属天佑医院中西医结合科

出　　处：《中华老年心脑血管病杂志》2019年第1期58-62,共5页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：国家自然科学基金(81500228;81270184);中央高校基本科研业务费青年教师专项资金(2042015kf0082)

摘　　要：目的探讨斑蝥素对血小板衍生生长因子BB(PDGF-BB)诱导的大鼠血管平滑肌细胞增殖和迁移的作用及机制。方法组织块贴壁法获取原代血管平滑肌细胞(vascular smooth muscular cells,VSMC),运用VSMC表型标志蛋白抗体免疫荧光染色鉴定细胞,PDGF-BB诱导细胞增殖和迁移,并使用不同浓度斑蝥素加以处理。将实验分为,对照组(A组); PDGF-BB组(B组); PDGF-BB+5μmol/L斑蝥素组(C组); PDGF-BB+10μmol/L斑蝥素组(D组)。采用CCK-8细胞计数试剂盒检测细胞活性和增殖情况。细胞划痕实验检测细胞迁移水平。Western blot检测基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)、总蛋白激酶B(t-Akt)、磷酸化蛋白激酶B(p-Akt)及平滑肌细胞特异性蛋白(α-smooth muscle actin,α-SM-actin)表达水平。结果与DMSO处理的细胞比较,不同剂量(0.625、1. 25、2. 5、5和10μmol/L)斑蝥素处理的细胞活性无统计学差异(P> 0. 05)。与A组比较,B组细胞增殖活性和细胞迁移率显著提高[2. 46±0. 19 vs 1. 49±0. 12,(88. 79±0. 61)%vs (42. 63±3. 93)%,P <0. 05];与B组比较,C组、D组细胞增殖活性和细胞迁移率显著降低[0. 91±0. 07 vs 2. 46±0. 19,0. 61±0. 04 vs 2. 46±0. 19,(39. 90±4. 30)%vs (88. 79±0. 61)%,(20. 33±6. 13)%vs (88. 79±0. 61)%,P <0. 05]。Western blot结果显示,与A组比较,B组MMP-9、p-Akt的表达水平显著增加,α-SM-actin的表达水平显著降低(P <0. 05);与B组比较,C组、D组MMP-9、p-Akt的表达水平显著降低,α-SM-actin的表达水平显著增加,差异有统计学意义(P <0. 05)。结论斑蝥素可显著抑制PDGF-BB诱导的VSMC增殖和迁移,其机制可能与下调p-Akt及MMP-9蛋白表达水平有关。Objective To study the mechanism of cantharidin underlying the proliferation and migration of vascular smooth muscular cells induced by PDGF-BB.Methods Primary vascular smooth muscular cells were isolated using tissue adherent method and identified with immunofluorescence staining of their phenotype marker protein antibodyα-smooth muscle actin.The proliferarion and migration of vascular smooth muscular cells were induced by PDGF-BB and conditioned in different cantharidin levels.The vascular smooth muscular cells were divided into control group(group A),PDGF-BB conditioned group(group B),PDGF-BB+5μmol/L cantharidin conditioned group(group C),PDGF-BB+10μmol/L cantharidin conditioned group(group D).The viability and proliferation of vascular smooth muscular cells were detected with a CCK-8 cell counting kit.The migration of VSMC was assayed in cell scratch test.The expressions of matrix metalloproteinase-9(MMP-9),t-Akt,p-Akt,α-smooth muscle actin were detected by Western blot.Results No significant difference was found in the viability of vascular smooth muscular cells conditioned in DMSO and different cantharidin doses(P >0.05).The proliferation activity and migration rate of vascular smooth muscular cells were significantly higher in group B than in group A and significantly lower in groups C and D than in group B(2.46±0.19 vs 1.49±0.12,88.79%±0.61%vs 42.63%±3.93%,P<0.05;0.91±0.07 vs 2.46±0.19,0.61±0.04 vs 2.46±0.19,39.90%±4.30%vs 88.79%±0.61%,20.33%±6.13%vs 88.79%±0.61%,P<0.05).Western blot showed that the expression levels of MMP-9 and p-Akt were significantly higher while those ofα-smooth muscle actin were significantly lower in group B than in group A(P<0.05).Conclusion Cantharidin can effectively inhibit the proliferation and migration of vascular smooth muscular cells induced by PDGF-BB by downregulating the expressions of p-AKT and MMP-9.

关 键 词：斑蝥素 受体 血小板源生长因子 肌细胞 平滑肌 细胞增殖 基质金属蛋白酶9 

分 类 号：R285[医药卫生—中药学]