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目的研究丹红注射液在共培养体系中通过人脐静脉内皮细胞(Ea.Hy926)对大鼠胸主动脉平滑肌细胞(A7r5)舒张的影响及其作用机制。方法以A7r5、Ea.Hy926为细胞模型,用Western blotting法检测丹红注射液对单独培养的A7r5细胞中肌球蛋白轻链(MLC)和磷酸化MLC(P-MLC)蛋白表达的影响;检测其对A7r5细胞内钙离子浓度的影响;用RT-PCR法检测A7r5细胞钙调蛋白(Ca M)m RNA表达的变化,MTT法观察其对重组人血小板源性生长因子-BB(PDGF-BB)诱导的A7r5细胞增殖的影响;最后用Transwell小室建立共培养体系,分别用Western blotting法和ELISA法检测丹红注射液对共培养体系中A7r5细胞MLC、P-MLC的蛋白表达以及环磷酸腺苷(c AMP)分泌的影响。结果丹红注射液(10μL/m L)能降低单独培养的A7r5细胞中P-MLC/MLC的值(P<0.05)和Ca M m RNA表达(P<0.01),对细胞内钙离子浓度和MLC蛋白表达无显著影响,也可降低共培养体系中A7r5细胞P-MLC/MLC值(P<0.05),并增加c AMP的分泌(P<0.01),此外还能抑制PDGF-BB诱导的A7r5细胞增殖(P<0.01)。结论丹红注射液能通过降低P-MLC/MLC值和Ca M的m RNA表达直接使平滑肌舒张,也能通过促进内皮细胞释放舒血管因子间接增加共培养体系中平滑肌细胞c AMP的分泌,使平滑肌细胞舒张。
Objective To investigate the effect of Danhong injection on the relaxation of rat aorta smooth muscle cells (A7r5) induced by human umbilical vein endothelial cells (Ea-Hy926) in co-culture system and its mechanism. Methods A7r5, Ea.Hy926 cell model was used to detect the effect of Danhong injection on the expression of myosin light chain (MLC) and phosphorylated MLC (P-MLC) protein in cultured A7r5 cells by Western blotting. (CaM) m RNA expression in A7r5 cells was detected by RT-PCR. The effect of recombinant human PDGF-BB (PDGF-BB) on the expression of CaM mRNA in A7r5 cells was observed by MTT assay. ) Induced proliferation of A7r5 cells. Finally, a co-culture system was established with Transwell chambers. The protein expression of MLC and P-MLC in A7r5 cells and the expression of cAMP in co-culture system were detected by Western blotting and ELISA respectively (C AMP) secretion. Results Danhong injection (10μL / m L) decreased the P-MLC / MLC (P <0.05) and CaM m RNA expression in A7r5 cells (P <0.01) (P <0.05), and increased the secretion of c AMP (P <0.01). In addition, it also inhibited the expression of A7r5 induced by PDGF-BB Cell proliferation (P <0.01). Conclusion Danhong injection can directly relax the smooth muscle by reducing the expression of P-MLC / MLC and CaM mRNA, and indirectly increase the c AMP secretion in smooth muscle cells of coculture system by promoting the release of vasodilators by endothelial cells. Smooth muscle cells relax.