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目的:观察雷帕霉素(RAPA)聚乳酸-羟基乙酸共聚物(PLGA)纳米微粒(RAPA-PLGA-NPs)对血管平滑肌细胞(VSMCs)增殖的抑制作用。方法:原代培养大鼠VSMCs,并采用α-肌动蛋白免疫组化染色进行鉴定;透射电镜观察VSMCs对RAPA-PLGA-NPs的摄取;分别用MTT法和Western blot法检测RAPA-PLGA-NPs对VSMCs增殖的抑制作用以及RAPA靶蛋白(mTOR)下游蛋白表达的影响,并同时以游离RAPA作为对照。结果:成功培养VSMCs并鉴定;透射电镜显示RAPA-PLGA-NPs可被VSMCs大量摄取于细胞质中;MTT结果显示,RAPA-PLGA-NPs(1,10,100 ng/mL)能浓度依赖性地抑制VSMCs增殖(均P<0.05),且1 ng/mL的RAPA-PLGA-NPs与10 ng/mL游离RAPA的抑制作用相似(P>0.05);Western blot结果显示,2个浓度(1,10 ng/mL)的RAPA-PLGA-NPs均能降低VSMCs中S6K1和4E-BP1磷酸化水平,且作用均较游离RAPA(10 ng/mL)明显。结论:RAPA-PLGA-NPs穿透力强,能迅速被细胞摄取,生物学效应明显强于其游离药物。
Objective: To observe the inhibitory effect of RAPA-PLGA nanoparticles (NPs) on the proliferation of vascular smooth muscle cells (VSMCs). Methods: Rat primary VSMCs were cultured and identified by α-actin immunohistochemical staining. The uptake of RAPA-PLGA-NPs by VSMCs was observed by transmission electron microscopy. The expressions of RAPA-PLGA-NPs On the proliferation of VSMCs and the expression of RAPA target protein (mTOR), and at the same time using free RAPA as control. Results: The VSMCs were successfully cultured and identified. Transmission electron microscopy showed that RAPA-PLGA-NPs could be taken up by the VSMCs in cytoplasm. MTT assay showed that RAPA-PLGA-NPs inhibited the proliferation of VSMCs in a concentration- (P <0.05). The inhibitory effect of RAPA-PLGA-NPs at 1 ng / mL and 10 ng / mL RAPA was similar (P> 0.05) ) RAPA-PLGA-NPs could reduce the phosphorylation of S6K1 and 4E-BP1 in VSMCs, and the effect was more obvious than that of free RAPA (10 ng / mL). CONCLUSION: RAPA-PLGA-NPs are highly penetrating and can be quickly taken up by the cells. The biological effects of RAPA-PLGA-NPs are stronger than that of free drugs.