论文部分内容阅读
目的:探讨靶向肝癌干细胞的紫杉醇纳米粒的制备及其对肝癌HepG2细胞（CD133阳性亚群占8%）和Huh-7细胞（CD133阳性亚群占65%）的效果。方法:应用单乳溶媒挥发法制备聚乳酸-聚羟基乙酸共聚物装载的紫杉醇纳米粒，采用1-乙基-3-（3-二甲基氨丙基）-碳化二亚胺/N-羟基琥珀酰亚胺（EDC/NHS）交联法制备由抗CD133抗体修饰的紫杉醇纳米粒，即靶向纳米粒。检测纳米粒的表征及理化特性（包封率和载药率、粒度分布、形态、体外释放）。将肝癌Huh-7和HepG2细胞与紫杉醇纳米粒或靶向纳米粒共培养，应用流式细胞术分析肝癌细胞对纳米粒的摄取和累积，并检测CD133阳性细胞比例，应用平板克隆形成实验分析细胞存活情况。结果:扫描电镜检测显示，靶向纳米粒的粒径为（429.26±41.53）nm，Zeta电位为-11.2 mV，具有球形形态和较高的包封率[（87.53±5.90）%]。流式细胞术检测显示，37 ℃时，Huh-7细胞靶向纳米粒组的荧光强度较紫杉醇纳米粒组荧光强度高（13 397±720比6 898±604），差异有统计学意义（n P0.05）。Huh-7细胞靶向纳米粒组CD133阳性细胞比例较紫杉醇纳米粒组低[（15.7±2.6）%比（54.9±7.4）%]，差异有统计学意义（n t=7.31，n P=0.008）；HepG2细胞两组间差异无统计学意义（n P>0.05）。平板克隆形成实验结果显示，靶向纳米粒作用后，各时间点Huh-7细胞存活率低于紫杉醇纳米粒作用后，差异有统计学意义（n F=5.56，n P=0.009）；紫杉醇纳米粒和靶向纳米粒作用后，HepG2细胞存活率差异无统计学意义（n F=1.19，n P=0.142）。n 结论:制备的靶向肝癌干细胞的纳米粒对肝癌Huh-7细胞有良好抑制作用。“,”Objective:To investigate the preparation of paclitaxel-loaded nanoparticles targeting liver cancer stem cells and their effects on liver cancer HepG2 cells (CD133 positive subset accounting for 8%) and Huh-7 cells (CD133 positive subset accounting for 65%).Methods:Poly (lactic-co-glycolic acid)-loaded paclitaxel nanoparticles were prepared by using emulsification-solvent evaporation method. Paclitaxel-loaded nanoparticles decorated with anti-CD133 antibody, called targeted nanoparticles, were prepared by using 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide/N-hydroxysuccinimide (EDC/NHS) cross-linking method. The manifestations and physicochemical characteristics of the nanoparticles including encapsulation efficiency, loading efficiency, particle size distribution, morphology and release in vitro were studied. Liver cancer Huh-7 and HepG2 cells accompanying with paclitaxel-loaded nanoparticles or targeted nanoparticles were cocultured. The uptake and accumulation of nanoparticles by liver cancer cells were analyzed by using flow cytometry, and positive cell proportion of CD133 was also detected. Cell survival was analyzed by using plate clonogenesis assay.Results:Scan electromicroscopy result showed particle size of targeted nanoparticles was (429.26±41.53) nm with zeta potential of -11.2 mV; targeted nanoparticles were possessed with spherical morphology and higher encapsulation efficiency [(87.53±5.90) %]. Flow cytometry showed that in Huh-7 cells at 37℃, the fluorescence intensity of targeted nanoparticles group (13 397±720) was higher than that of paclitaxel-loaded nanoparticles group (6 898±604), and the difference was statistically significant (n P 0.05). CD133 positive cell proportion of Huh-7 cells in targeted nanoparticles group [(15.7±2.6)%] was lower than that in paclitaxel-loaded nanoparticles group [(54.9±7.4)%], and the difference was statistically significant (n t = 7.31, n P = 0.008); there was no statistical difference of HepG2 cells between the two goups (n P > 0.05). Plate clonogenesis assay showed that the cell survival rate of Huh-7 cells in targeted nanoparticles group was lower than that in paclitaxel-loaded nanoparticles group at different time points, and the difference was statistically significant ( n F = 5.56, n P = 0.009); but there was no statistically significant difference in HepG2 cell survival rate between the two groups (n F = 1. 19, n P = 0.142).n Conclusion:Prepared nanoparticles targeting liver cancer stem cells have a good inhibitory effect on liver cancer Huh-7 cells.