目的联合体内外实验,探讨钝顶螺旋藻藻蓝蛋白(C-PC)抑制血管平滑肌细胞过度增殖、血管损伤后内膜增生和管腔狭窄的作用和机制。方法培养大鼠胸主动脉血管平滑肌细胞,用不同浓度的藻蓝蛋白进行干预,用氚标记胸腺嘧啶核苷(3H-TdR)参入率检测血管平滑肌细胞DNA合成和增殖状态,用Western blot和图像分析方法检测细胞周期蛋白依赖激酶抑制因子P21、P27的表达。构建大鼠胸主动脉球囊损伤后再狭窄动物模型,用藻蓝蛋白进行灌胃干预,术后14d取胸主动脉应用HE染色、免疫组化和计算机图像分析仪进行形态学、肌动蛋白(SM-α-actin)、增殖细胞核抗原(PCNA)、P21、P27表达水平检测。结果胎牛血清诱导的血管平滑肌细胞增殖活跃,P21、P27低表达;藻蓝蛋白明显促进P21、P27蛋白表达,明显抑制胎牛血清诱导的血管平滑肌细胞增殖和DNA合成。大鼠胸主动脉球囊损伤后,平滑肌细胞向内膜迁移、过度增殖、形成明显的新生内膜,导致管腔狭窄,SM-α-actin免疫组化证实新生内膜富含平滑肌细胞,PCNA高表达的同时P21、P27处于低表达水平;藻蓝蛋白干预后明显促进P21、P27基因表达,下调PCNA表达,抑制新生内膜形成、病理性血管重构,从而明显抑制管腔狭窄。结论钝顶螺旋藻藻蓝蛋白具有抑制血管平滑肌细胞过度增殖、血管损伤后管腔狭窄的作用,其机制与促进细胞周期关键调节因子P21、P27基因表达有关。
Aim To investigate the effects of C-phycocyanin(C-PC) from Spirulina Platensis on over proliferation of vascular smooth muscle cells(VSMCs),intimal hyperplasia and luminal stenosis after vascular injury.Methods The VSMCs of rat thoracic aorta were cultured and treated with different concentrations of Cphycocyanin.DNA synthesis and proliferation state of VSMCs were determinated with tritiated thymidine(3H-TdR) incorporation,the expression of of cyclindependent kinase inhibitors(P21 and P27) was detected with Western blot and image analysis.Rat model with restenosis of thoracic aorta after balloon damage was constructed.The rats received intragastric administration of C-phycocyanin for 14 days,and the thoracic aorta were taken out.HE staining,immunohistochemistry and image analysis computer system were applied to measure the vessel morphology,SM-α-actin,proliferating cell nuclear antigen(PCNA),and protein expression of P21 and P27.Results Active proliferation of VSMCs and low expression of P21 and P27 were examined after VSMCs were induced by fetal bovine serum(FBS).C-phycocyanin could significantly inhibit VSMCs proliferation and DNA synthesis,and meanwhile up-regulated quantities of P21 and P27 protein.In rats with balloon injury,C-phycocyanin group had an outstandingly smaller noeintima / media area ratio,larger internal or external elastic lamina area and luminal area;and C-phycocyanin promoted the expression of P21 and P27.Conclusions C-phycocyanin can inhibit the over proliferation of VSMCs and luminal narrowing after aortic balloon injury.The mechanism is probably that C-PC can promote the expression of key regulatory factors P21 and P27.