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目的观察软脉灵药物血清对血小板源性生长因子(PDGF)诱导的血管平滑肌细胞(VSMC)骨架蛋白α肌动蛋白和调节蛋白丝切蛋白(cofilin)重排的影响。方法组织块贴壁法培养VSMC,α肌动蛋白单克隆抗体鉴定细胞,间隔12h灌胃5次后制备SD大鼠软脉灵药物血清;将VSMC按照如下方式进行处理:未作任何处理(空白对照组)、加入PDGF(10μg/L,PDGF组)、10%正常血清+PDGF(正常血清组)、10%软脉灵药物血清+PDGF(软脉灵药物血清组)和阿托伐他汀+PDGF(Ator组)。使用免疫荧光技术对丝切蛋白和α肌动蛋白进行染色,激光共聚焦显微镜下观察细胞骨架蛋白的重排情况,并通过共聚焦显微镜软件计算丝切蛋白的荧光强度变化。结果PDGF刺激后,细胞呈梭形,细胞骨架重排,细胞浆中出现应力纤维且排列集中;丝切蛋白表达增强(空白对照组32.05±6.10比PDGF组77.68±13.15,P<0.01),且和微丝“共位”于胞质和板状伪足处,沿细胞膜边缘呈线状分布。10%软脉灵药物血清干预1h后,细胞微丝无集中排列,应力纤维减少且排列松散,丝切蛋白表达减弱(软脉灵药物血清组61.15±6.23比PDGF组77.68±13.15,P<0.01;软脉灵药物血清组61.15±6.23比Ator组51.85±10.59,P=0.036),板状伪足减少且只有少量微丝和丝切蛋白分布。结论 PDGF可增强血管平滑肌细胞骨架蛋白丝切蛋白的表达;软脉灵药物血清可抑制细胞骨架调节蛋白丝切蛋白的表达和在胞质中重排,抑制PDGF诱导的VSMC形态变化。
Objective To observe the influence of Ruanmailing drug serum on the rearrangement of actin and cofilin of vascular smooth muscle cells (VSMCs) induced by platelet derived growth factor (PDGF). Methods The VSMCs were cultured by adherence with tissue blocks, and the cells were identified by α-actin monoclonal antibody. The rats were treated with gavage for 5 times at 12h intervals. The VSMCs were treated as follows: without any treatment Control group), PDGF (10μg / L, PDGF group), 10% normal serum + PDGF (normal serum group), 10% Rimaxan serum + PDGF and atorvastatin + PDGF (Ator group). Ficoll and α-actin were stained by immunofluorescence technique. The rearrangement of cytoskeletal proteins was observed under confocal laser scanning microscope. The fluorescence intensity of cofilin was calculated by confocal microscopy. Results After stimulation with PDGF, the cells were spindle-shaped and cytoskeleton rearranged. The stress fibers appeared in the cytoplasm and arranged in a concentrated manner. The expression of cofilin was increased in the control group (32.05 ± 6.10 in the blank control group and 77.68 ± 13.15 in the PDGF group, P <0.01) And Microfiber “common place ” in the cytoplasm and plate-like pseudopodia, along the cell membrane edge was linear distribution. After treated with 10% Ruanmailing serum for 1h, the fibroblasts were not arranged in a centralized manner, the stress fibers were reduced and loosely arranged, and the expression of cofilin was decreased (Ruanmailing serum 61.15 ± 6.23 compared with PDGF 77.68 ± 13.15, P <0.01 ; Ruanmailing drug serum group 61.15 ± 6.23 than the Ator group 51.85 ± 10.59, P = 0.036), platelet pseudopodia decreased and only a small amount of microfilament and cofilin distribution. Conclusion PDGF can enhance the expression of skeletal cortical protein of vascular smooth muscle cells. Ruanmailing serum can inhibit the expression of cytoskeleton regulatory protein cofilin and rearrangement in the cytoplasm, and inhibit PDGF-induced VSMC morphological changes.