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目的观察雷帕霉素(RPM)及其乳酸-乙醇酸共聚物(PLGA)形成的载药纳米粒子(RPM-PLGA-NPs)在不同浓度和作用时间下对离体培养人脐动脉平滑肌细胞(HUASMCs)细胞凋亡及调控基因bcl-2、p27kip1表达的影响。方法离体培养HUASMCs细胞并分别予不同浓度的RPM、RPM-PLGA-NPs作用12和24 h,设立生理盐水及空白PLGANPs对照组,免疫组织化学方法检测p27kip1与bcl-2表达阳性率和表达水平的差异,采用流式细胞仪、DNA片段琼脂糖凝胶电泳及末端原位标记法检测各组HUASMCs凋亡及细胞凋亡率,噻唑蓝比色法观察不同浓度RPM和RPM-PLGA-NPs对HUASMCs存活率的影响。结果 RPM及RPM-PLGA-NPs组抑制HUASMCs增殖并呈剂量依赖关系,HUASMCs DNA电泳呈梯度条带阳性。流式细胞仪检测RPM 100 ng/ml和RPM-PLGA-NPs 500 ng/ml组的细胞凋亡百分率分别为(45.45±2.36)%和(35.04±5.64)%,显著高于对照组的(2.60±0.95)%(P<0.01)。末端原位标记法检测高剂量干预组的24 h凋亡指数显著高于12 h组(P<0.05,P<0.01)。RPM 100 ng/ml组和RPM-PLGA-NPs 500 ng/ml组的p27kip1蛋白阳性表达指数(PEI)分别为(0.178±0.077)%和(0.192±0.052)%,显著高于对照组的(0.101±0.035)%(P<0.05)。Spearman秩相关检验显示RPM及RPM-PLGA-NPs组凋亡指数值与p27kip1的PEI无相关性。RPM-PLGA-NPs 50 ng/ml及RPM 10 ng/ml组bcl-2的PEI分别为(6.44±1.31)%和(5.49±1.06)%,显著高于对照组的(1.84±0.47)%和(2.06±0.53)%(P<0.05)。结论 RPM及RPM-PLGA-NPs上调离体培养的HUASMCs的p27kip1表达、促进细胞凋亡,并无下调抗细胞凋亡基因bcl-2表达的作用。相当载药量的RPM-PLGA-NPs较RPM促进血管平滑肌细胞凋亡的作用更明显,但与p27kip1表达水平无线性相关。
OBJECTIVE: To investigate the effect of RPM-PLGA-NPs on the proliferation of human umbilical artery smooth muscle cells (VSMCs) cultured in vitro at different concentrations and duration of action (RPMs) and their lactic acid-glycolic acid copolymer (PLGA) HUASMCs) and the expression of bcl-2 and p27kip1. Methods HUASMCs were cultured in vitro and treated with different concentrations of RPM and RPM-PLGA-NPs for 12 and 24 h, respectively. Normal saline and PLGANPs control group were established. Immunohistochemistry was used to detect the expression of p27kip1 and bcl-2 , The apoptosis and apoptosis rate of HUASMCs in each group were detected by flow cytometry, DNA agarose gel electrophoresis and end-in-situ labeling method. The different concentrations of RPM and RPM-PLGA-NPs The impact of HUASMCs survival rate. Results RPM and RPM-PLGA-NPs inhibited the proliferation of HUASMCs in a dose-dependent manner. The HUASMCs showed positive gradient DNA bands. The percentage of apoptotic cells in RPM 100 ng / ml and 500 ng / ml RPM-PLGA-NPs groups was (45.45 ± 2.36)% and (35.04 ± 5.64)%, respectively, which was significantly higher than that in control group ± 0.95)% (P <0.01). The 24 h apoptosis index of the high-dose intervention group was significantly higher than that of the 12 h group (P <0.05, P <0.01). The positive expression rates of p27kip1 protein in RPM 100 ng / ml group and RPM-PLGA-NPs 500 ng / ml group were (0.178 ± 0.077)% and (0.192 ± 0.052)%, respectively, which were significantly higher than those in control group ± 0.035)% (P <0.05). Spearman rank correlation test showed no correlation between the apoptotic index of RPM and RPM-PLGA-NPs group and PEI of p27kip1. The PEI of RPM-PLGA-NPs in 50 ng / ml and RPM 10 ng / ml groups was (6.44 ± 1.31)% and (5.49 ± 1.06)%, which was significantly higher than that in control group (1.84 ± 0.47)% and (2.06 ± 0.53)% (P <0.05). Conclusion RPM and RPM-PLGA-NPs upregulate the expression of p27kip1 in HUASMCs in vitro, and promote the apoptosis of HUASMCs without down-regulating the expression of anti-apoptotic gene bcl-2. The RPM-PLGA-NPs with comparable drug loading were more effective than RPM in promoting vascular smooth muscle cell apoptosis, but not with the p27kip1 expression level.