摘要
[目的]探讨趋化因子12(chemokine 12,CXCL12)/趋化因子受体4(chemokine receptor 4,CXCR4)生物轴在清心饮含药血清抑制柯萨奇B3病毒(Coxsackie virus B3,CVB3)感染后心脏微血管内皮细胞(cardiac microvascular endothelial cells,CMVECs)发生内皮间充质转分化(endothelial-mesenchymal transition,EndMT)过程中的作用。[方法]取生长状态良好的CMVECs设计为正常对照组、模型组、CXCR4拮抗剂组、清心饮组和清心饮+CXCR4拮抗剂组。除正常对照组用含10%胎牛血清(fetal bovine serum,FBS)的杜尔伯克改良伊格尔培养基(Dulbecco’s modification of Eaglews medium,DMEM)培养外,其余各组CMVECs先用CVB3培养基感染2 h,后弃去CVB3培养基,改为普通培养。通过细胞计数试剂(cell counting kit-8,CCK8)检测各组细胞活力,免疫荧光(immunofluorescence,IF)、免疫印迹和实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)等实验方法检测内皮细胞标志物血小板-内皮细胞黏附分子(platelet endothelial cell adhesion molecule-1,PECAM-1/CD31)、间充质细胞标志物α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、CXCL12和CXCR4的蛋白及基因表达。[结果]与正常对照组比较,模型组CD31表达下降(P<0.01),α-SMA、CXCL12和CXCR4表达增强(P<0.01)。与模型组比较,CXCR4拮抗剂组、清心饮组CD31表达增强(P<0.01),α-SMA和CXCR4表达下降(P<0.01),CXCR4拮抗剂组CXCL12表达差异无统计学意义(P>0.05),清心饮组CXCL12表达下降(P<0.05)。与清心饮组比较,清心饮+CXCR4拮抗剂组CD31的表达增强(P<0.05),α-SMA、CXCL12和CXCR4表达下降(P<0.01,P<0.05)。[结论] CXCL12/CXCR4生物轴参与了CVB3感染后CMVECs发生EndMT的过程,拮抗CXCL12/CXCR4生物轴能够明显增强清心饮对该过程的抑制。
[Objective] To explore the intervention effect of Qingxinyin pharmacologic serum in inhibiting the endothelial-mesenchymal transition(EndMT) in Coxsackie virus B3(CVB3) infected cardiac microvascular endothelial cells(CMVECs) via regulating chemokine 12/chemokine receptor 4(CXCL12/CXCR4) biological axis. [Methods] CMVECs in good growth status were taken, and according to the experimental purpose, normal control group, model group, CXCR4 antagonist group, Qingxinyin group and Qingxinyin +CXCR4antagonist group were designed. Except for normal control group, which was cultured with Dulbecco’s modification of Eagle’s medium(DMEM) containing 10% fetal bovine serum(FBS), the other groups were infected CMVECs with CVB3 culture medium for 2 hours,and abandoned CVB3 culture medium, and then continued to normal culture. The cell activity of each group was performed by cell counting kit-8(CCK8). Theproteinandgeneexpressions of the endothelial cell marker plateletendothelialcelladhesionmolecule-1(PECAM-1/CD31), the mesenchymal cell marker α-smooth muscle actin(α-SMA), CXCL12 and CXCR4 were detected with immunofluorescence(IF),Western blot and Real-time quantitative polymerase chain reaction(Real-time qPCR). [Results] Compared with normal control group, the expression of CD31 was decreased(P<0.01), and the expressions of α-SMA, CXCL12 and CXCR4 were increased in model group(P<0.01).Compared with model group, the expression of CD31 was increased(P<0.01), and the expressions of α-SMA and CXCR4 were reduced in CXCR4 antagonist group and Qingxinyin group(P<0.01), there was no statistically significant expression of CXCL12 in CXCR4 antagonist group(P>0.05), the expression of CXCL12 was decreased in Qingxinyin group(P<0.05). Compared with Qingxinyin group, the expression of CD31 was increased(P <0.05), and the expressions of α-SMA, CXCL12 and CXCR4 were reduced in Qingxinyin +CXCR4 antagonist group(P <0.01, P <0.05). [Conclusion] The CXCL12/CXCR4 biological axis is involved in the occurrence of EndMT in CVB3-infected CMVEC
作者
代楚行
陈俊
王壹民
刘强
DAI Chuxing;CHEN Jun;WANG Yimin(Zhejiang Chinese Medical University,Hangzhou(310053),China)
出处
《浙江中医药大学学报》
CAS
2022年第7期699-707,727,共10页
Journal of Zhejiang Chinese Medical University
基金
国家自然科学基金面上项目(81873247)。
