鬼臼毒素纳米脂质载体的制备及体外对HPV感染的永生化人宫颈上皮细胞的作用  

Preparation of podophyllotoxin nanostructured lipid carriers and its effects on immortalized human cervical epithelial cells with HPV infection in vitro

作　　者：种树彬[1] 曾抗[1] 李国锋[2] 任非[2] 朱晓亮[1] 周金洁[1] 

机构地区：[1]南方医科大学南方医院皮肤科,广东广州510515 [2]南方医科大学南方医院药学部,广东广州510515

出　　处：《南方医科大学学报》2011年第6期1023-1028,共6页Journal of Southern Medical University

基　　金：广东省科技计划项目(2007B031003006)

摘　　要：目的研究鬼臼毒素纳米脂质载体（POD-NLC）的体外对HPV感染的永生化人宫颈上皮细胞的作用。方法采用乳化蒸发法制备POD-NLC,分别应用扫描电镜、粒径仪、高效液相法等方法研究其表征,并观察其稳定性。分别以不同浓度（0.00011μg/ml）的POD-NLC和鬼臼毒素（POD）处理H8细胞,采用MTT法检测细胞的增殖活性,以荧光显微镜观察细胞的形态变化,流式细胞术（FCM）检测其凋亡率和细胞周期。结果①制备的POD-NLC扫描电镜下呈球形,有较好的稳定性,粒径为（85.6±10.25）nm,电位为（26.2±4.1）mV,包封率为（88.56±3.1）%;②POD-NLC能较强地抑制H8细胞的增殖,其抑制作用呈现时间和剂量依赖性;POD-NLC和POD作用72h对H8细胞的抑制率最高分别为95.8%、65.6%,半数抑制浓度（IC50）分别为0.015、0.13μg/ml;空白NLC对H8细胞的增殖无影响;③浓度为0.01μg/ml的POD-NLC和POD分别处理H8细胞48h后,H8细胞的凋亡率分别为（88.30±4.28）%和（59.95±3.26）%;荧光显微镜观察,均出现了核固缩、染色质高度凝聚、凋亡小体等典型的凋亡形态改变;与空白对照组相比,POD-NLC组和POD组G2/M期细胞比例明显增多,S期细胞比例无明显变化,G0/G1期细胞比例明显减少,但两组间细胞周期时相比例的差异无统计学意义（P〉0.05）。结论该制备工艺可行;与POD相比,POD-NLC对H8细胞具有更强的增殖抑制和凋亡诱导效果,可使细胞阻滞于G2/M期。Objective To study the effect of podophyllotoxin nanostructured lipid carriers （POD-NLC） on immortalized human cervical epithelial cells （H8） infected with HPV in vitro. Methods POD-NLC was prepared by emulsion evaporation method and characterized using transmission electron microscopy, Zetasizer analyzer and high-performance liquid chromatography （HPLC）. H8 cells were treated with different concentrations （0.0001-1 μg/ml） of POD-NLC, free POD, or blank nanostructured lipid carriers （NLC）, and the cell proliferation was assessed using MTT assay to evaluate the cytotoxic effects. The changes of cell morphology were observed using fluorescence microscopy, and the cell cycle changes and cell apoptosis were analyzed using flow cytometry. Results POD-NLC showed a spherical or elliptical shape with good stability in vitro. The average particle size of POD-NLC was 85.6±10.25 nm, with a Zeta potential of 26.2±4.1 mV and entrapment efficiency of POD of （88.56±3.1）%. POD-NLC caused a significant inhibition of H8 cell proliferation in a concentration- and time-dependent manner. At an equivalent concentration, POD-NLC produced a stronger inhibitory effect on cell proliferation than POD. The inhibition rate of H8 cells after a 48-h exposure to POD-NLC and POD reached 95.8% and 65.6%, respectively, and at the highest concentration of 1 μg/ml, the IC50 of POD-NLC and POD was 0.015 μg/ml and 0.13 μg/ml, respectively. Blank NLC did not obviously affect the proliferation of H8 cells. POD-NLC and POD both caused obvious increases in G2/M phase cell percentages and induced typical apoptotic changes of the cells, and their effects were comparable （P〉0.05）. Conclusions Compared with POD, POD-NLC has more potent effect in inhibiting H8 cell proliferation and inducing cell apoptosis, suggesting its potential in the treatment of cervical HPV infection

关 键 词：鬼臼毒素 纳米结构脂质载体 细胞凋亡 永生化人宫颈上皮细胞 

分 类 号：R283[医药卫生—中药学] R285[医药卫生—中医学]