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目的评估本实验室自制包载雷帕霉素(RPM)的聚乳酸-聚乙醇酸共聚物(PLGA)纳米粒子(NPs)对离体培养的人脐动脉平滑肌细胞(HUASMC)细胞周期时相、p27蛋白表达和细胞增殖的影响。方法按不同浓度RPM-PLGA NPs设定药物作用组,并设立RPM组、PLGA组和M231培养基及平滑肌细胞生长添加剂(M231-SMGs)组作为对照。采用细胞免疫组织化学染色比较不同处理组HUASMC p27蛋白表达阳性率和表达水平的差异,流式细胞技术评价各处理组对HUASMC细胞周期时相的影响,噻唑蓝(MTT)比色法观察不同浓度RPM和RPM-PLGA NPs对HUASMC存活率的影响。结果10μg/L及以上浓度的RPM和50μg/L及以上浓度的RPM-PLGA NPs可明显抑制HUA-smc生长,并呈浓度依赖性。细胞计数绘制生长-时间曲线显示,100μg/L RPM和500μg/L RPM-PLGA NPs作用于HUASMC 24 h后细胞计数值明显低于M231-SMGs对照组;单纯PLGA NPs对细胞生长无明显影响;与PLGA组和M231-SMGs培养基对照组相比,RPM组和RPM-PLGA NPs组G_0/G_1期细胞比例明显增多,S期及G_2/M期细胞比例明显减少(P均<0.01),但两组间细胞周期时相比例差异无统计学意义(P>0.05)。细胞免疫组织化学染色结果显示,100μg/LRPM和500μg/L RPM-PLGA NPs组的HUASMC p27蛋白表达阳性率和表达水平与对照组相比差异无统计学意义(P>0.05),但均较PLGA组和M231-SMGs培养基组明显增加(P<0.01)。结论RPM-PLGA NPs抑制体外培养的HUASMC生长效果与RPM相似,并可显著抑制体外培养HUASMC p27蛋白表达,阻抑其细胞周期进程于G_1/S期而抑制其细胞增殖。
Objective To evaluate the effect of self-made polylactic acid-polyglycolic acid copolymer (PLGA) nanoparticles coated with rapamycin (RPM) on the cell cycle phases of human umbilical artery smooth muscle cells (HUASMC) cultured in vitro, p27 protein expression and cell proliferation. Methods The drug group was set up according to different concentrations of RPM-PLGA NPs. RPM group, PLGA group, M231 medium and M231-SMGs group were set as the control group. The positive rate and expression level of p27 protein in HUASMC from different treatment groups were compared by immunohistochemical staining. The effect of each treatment group on the cell cycle phase was evaluated by flow cytometry. MTT colorimetry was used to observe the expression of p27 protein, Effect of RPM and RPM-PLGA NPs on HUASMC Survival. Results RPM of 10μg / L and above and RPM-PLGA NPs of 50μg / L and above significantly inhibited HUA-smc growth in a concentration-dependent manner. Cell growth curve showed that the cell counts of HUASMCs treated with 100μg / L RPM and 500μg / L RPM-PLGA NPs for 24 h were significantly lower than that of M231-SMGs control group. PLGA NPs alone had no significant effect on cell growth. Compared with the control group, the proportion of G 0 / G 1 phase in RPM group and RPM-PLGA NPs group was significantly increased, while the proportion of S phase and G 2 / M phase group was significantly decreased in both PLGA group and M231-SMGs medium group (P <0.01) There was no significant difference in the proportion of cell cycle between the two groups (P> 0.05). Immunocytochemical staining showed that there was no significant difference in the positive rate and expression level of p27 protein in HUASMCs between 100μg / L RPM and 500μg / L RPM-PLGA NPs groups compared with control group (P> 0.05) Group and M231-SMGs medium group increased significantly (P <0.01). Conclusion RPM-PLGA NPs can inhibit the growth of HUASMC in vitro in a similar manner to that of RPM, and significantly inhibit the expression of HUASMC p27 protein in vitro and inhibit its cell cycle progression at G 1 / S phase and inhibit its proliferation.