Institute of Mechanobiology and Medical Engineering,Shanghai Jiao Tong University
目的 探讨周期性张应变力学刺激对血管平滑肌细胞（vascular smooth muscle cells，VSMCs）与血小板微体（platelet-delivered microparticles, PMPs）黏附能力的影响，以及黏附的PMPs对VSMCs自噬的调控作用。方法 应用 FX-5000T 张应变加载系统，对体外培养的VSMCs 施加 5% 幅度的生理性张应变和15% 幅度的高张应变；应用流式细胞术检测不同张应变作用的VSMCs 与 PMPs 的黏附；免疫荧光检测PMPs刺激24 h后自噬标志分子微管相关蛋白轻链3（autophagy microtubule associated protein light chain 3，LC3）的表达水平；Western blotting 检测PMPs刺激24 h后VSMCs自噬相关蛋白（autophagy related protein，Atg）的表达水平。结果 与5% 生理性张应变加载相比，15% 高张应变加载24 h能显著增强VSMCs与PMPs的黏附水平，提示高张应变促进PMPs与VSMCs的黏附。免疫荧光和Western blotting结果显示PMPs刺激可显著上升VSMCs中自噬标志蛋白LC3表达，同时Western blotting 检测到PMPs刺激后Atg5、Atg7和分子量为55 KDa的Atg12蛋白表达水平显著上升。结论 高张应变可以促进VSMCs黏附PMPs，黏附的PMPs可能通过增加Atg5、Atg7、Atg12、LC3表达，从而增强VSMCs自噬。探讨张应变力学刺激调控VSMCs功能的力学生物学分子机制，对于揭示血管生理稳态维持和血管病理重建的分子机制具有一定意义，并为寻找内膜损伤血管重建疾病诊断、临床治疗和疗效评价的潜在靶点提供了新视角。
Objective To investigate the effect of cyclic stretch on adhesion of vascular smooth muscle cells (VSMCs) with platelet-delivered microparticles (PMPs), and the role of PMPs in VSMC autophagy. Methods Using FX-5000T cyclic stretch loading system, cyclic stretch with the magnitude of 5% (mimics physiological mechanical stretch) or 15% (mimics pathological mechanical stretch) was subjected to VSMCs in vitro; using flow cytometry the adhesion of PMPs in VSMCs was detected. Immunofluorescence was used to detect the expression of autophagy microtubule associated protein light chain 3 (LC3) after 24 h stimulation with PMPs. Western blotting was used to detect the expression of autophagy related protein (Atg) in VSMCs after 24 h stimulation by PMPs. Results Compared with 5% cyclic stretch, 15% cyclic stretch significantly increased the adhesion ability of VSMCs with PMPs. Immunofluorescence snd western blotting revealed that PMPs stimulation significantly increased the expression of autophagy marker protein LC3 in VSMCs. Furthermore, the protein expressions ofAtg5, Atg7 and Atg12 (with 55 KDa molecular weight buth not 16 KDa) were all significantly increased in VSMCs stimulated with PMPs. Conclusion High cyclic stretch may enhance the autophagy of VSMCs by promoting the adhesion of PMPs, which subsequently increase the expressions of Atg5, Atg7, Atg12 and LC3. The investigation on mechanobiological mechanisms of VSMC autophagy induced by cyclic stretch may contribute to further understanding the vascular homeostasis and vascular remodeling, and may provide new potential targets for the clinical diagnosis, therapy, and evaluation of intimal injury during vascular reconstruction diseases.