包载雷帕霉素的乳酸-乙醇酸共聚物纳米粒子对人脐动脉平滑肌细胞bcl-2、p27^(kip1)表达及细胞凋亡的影响  

Effects of Rapamycin and Rapamycin-loaded Poly( lactic-co-glycolic) Acid Nanoparticles on Apoptosis and Expression of bcl-2 and p27^(kip1) Proteins of Human Umbilical Arterial Vascular Smooth Muscle Cell

作　　者：苗立夫 崔永亮 尹燕平 陈莲凤[2] 张华[3] 刘佩毛[3] 朱文玲[2] 宋存先[4] 杨菁[4] 

机构地区：[1]清华大学第一附属医院心脏中心,北京100016 [2]中国医学科学院北京协和医学院北京协和医院心脏内科,北京100730 [3]中国医学科学院北京协和医学院基础医学研究所病理室,北京100730 [4]中国医学科学院北京协和医学院生物医学工程所天津市生物材料重点实验室,天津300192

出　　处：《中国医学科学院学报》2015年第6期633-640,共8页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金(30240021);清华-裕元医学科学研究基金(20240000556;20240000520)~~

摘　　要：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 investgate the effects of rapamycin（ RPM） and RPM-loaded poly（ lacticco-glycolic） acid（ PLGA） nanoparticles（ NPs） on the apoptosis of human umbilical arterial vascular smooth muscle cells（ HUASMCs） in vitro and expression of bcl-2 and p27kip1 protein. Methods HUASMCs were cultured in vitro and divided to RPM and RPM-PLGA-NPs groups treated at 3 different concentration by 12 and 24 hours,with M231- smooth muscle growth supplements medium and null-PLGA-NPs treated groups as controlled.The apoptosis of HUASMCs was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining and flow cytometry. The expressions of bcl-2 and p27kip1 were detected by streptacidin / peroxidase immunohistochemical method. The effect on cellular proliferation was assessed by 3-（ 4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromidecolorimetry. Results The proliferation of HUASMCs was inhibited by RPM and RPM-PLGA-NPs in a dose-dependent manner. DNA electrophoresis showed DNA ladder in RPM and RPM-PLGA-NPs groups and classical scalar strips in control groups. The apoptotic indexes of RPM 100 ng / ml group and RPM-PLGA-NPs 500 ng / ml group detected by flow cytometry were（ 45. 45 ±2. 36） % and（ 35. 04 ± 5. 64） %,respectively,which were significantly higher than that of M231-smooth muscle growth supplements control group [（ 2. 60 ± 0. 95） %,all P〈0. 01]. The apoptotic indexes of groups incubated with RPM and RPM-PLGA-NPs for 24 hours were significantly higher than those of groups which incubated for 12 hours（ P〈0. 05,P〈0. 01）. The positive expression indexes（ PEI） of p27kip1 and bcl-2 protein were higher in RPM and RPM-PLGA-NPs groups than that of control groups. The Spearman＇s rank correlation coefficient test showed that there was no significant correlation between the PEI of p27kip1 and the apoptotic indexes in the RPM group and RPM-PLGA-NPs group（ P〉0. 05）. Conclusions Rapamycin-loaded PLGA nanoparticles and r

关 键 词：雷帕霉素 乳酸-乙醇酸共聚物 纳米粒子 细胞凋亡 人脐动脉平滑肌细胞 

分 类 号：R318.08[医药卫生—生物医学工程] R972.6[医药卫生—基础医学]