中文摘要：

目的本研究采用叶酸靶向化的聚乙烯亚胺包被的磁性纳米复合物（Fa-PEISPIO）携带HIF-1α-si RNA真核表达质粒转染CDl33＋Hep3B肝癌干细胞,观察其对肝癌干细胞增殖能力的抑制效果并探讨其起效机制。方法采用CD133-PE荧光抗体流式分选Hep3B细胞中的CD133阳性细胞。采用流式细胞术检测分选前后细胞CDl33表达量。将Fa-PEI-SPIO纳米复合物与含HIF-1α小干扰RNA（HIF-1α-si RNA）、阴性对照（NC）-si RNA的真核表达质粒复合,分别形成Fa-PEI-SPIO/HIF-1α-si RNA和Fa-PEI-SPIO/NCsi RNA两种靶向化纳米复合物。分别应用两种纳米复合物转染CDl33＋Hep3B肝癌干细胞,建立Fa-PEI-SPIO/HIF-1α-si RNA（FH）组和Fa-PEI-SPIO/NC-si RNA（FN）组,另设立未转染对照（CT）组。Western-blot法检测各组细胞的HIF-1α和Cyclin D1蛋白表达。平板克隆形成实验检测各组细胞的克隆形成能力。流式细胞术检测各组细胞的细胞周期。CD133阳性细胞比例、蛋白平均相对表达量、有效克隆形成数量和细胞周期等实验数据以x±s表示,3组间比较采用单因素方差分析,两两比较采用LSD-t检验。结果分选前Hep3B肝癌细胞检测CD133阳性率为（2.95±0.04）﹪,分选后CDl33阳性率增加到（92.59±0.05）﹪,差异有统计学意义（t=34.88,P=0.00）。FH组CDl33＋Hep3B肝癌干细胞HIF-1α、cyclin D1蛋白平均相对表达量分别为0.11±0.02、0.38±0.03,CT组分别为0.38±0.07、0.59±0.05,差异具有统计学意义（LSD-t=15.88,23.75;P=0.01,0.00）。FH组和CT组CDl33＋Hep3B肝癌干细胞的有效克隆数量为分别为（25.92±5.22）个/孔和（364.00±13.93）个/孔,FH组的有效克隆数量明显低于CT组（LSD-t=-37.67,P=0.00）。与CT组相比,FH组细胞G0/G1期的细胞数量明显增大,出现了明显的G0/G1期阻滞,S期和G2/M期细胞数量明显降低,分裂活跃性较CT组降低。结论纳米复合物Fa-PEI-SPIO可高效负载HIF-1α-si RNA真核表达质粒抑制细胞内HIF-1α

英文摘要：

Objective In this study, folate-targeted polyethyleneimine coated magnetic nanocomposites（Fa-PEI-SPIO）carrying the HIF-1α-si RNA eukaryotic expression plasmid was transfected into CDl33＋ Hep3 B liver cancer stem cells, the effect on cell proliferation and its mechanism was observed on liver cancer stem cells. Methods CD133 positive Hep3 B cells were sorted by CD133-PE fluorescent antibody. Flow cytometry was used to detect the expression of CDl33 before and after sorting. The Fa-PEI-SPIO/ HIF-1α-si RNA and Fa-PEISPIO/ NC-si RNA compounds were producedrespectively and transfected into. CDl33＋ Hep3 B cells respectively. Expression of cyclin D1 and HIF-1α protein in the cells of each group were detected by western-blot assay. Clone formation ability of the cells in the experimental group was detected by plate clone formation assay. Cell cycle was detected by flow cytometry. The proportion of CD133 positive cells, the average relative expression amount of protein, the number of effective clones and the cell cycle were compared with the experimental data of x ± s, the 3 groups were compared by using single factor analysis of variance, and paired group was compared with LSD-t test. Results The positive rate of CD133 before sortting was（2.95 ± 0.04） ﹪ and the positive rate increased to（92.59 ± 0.05） ﹪（t = 34.88, P = 0.00）. The average relative expression of cyclin D1 and HIF-1 protein in FH and CT group were 0.11 ± 0.02, 0.38 ± 0.03 and 0.38 ± 0.07, 0.59 ± 0.05 respectively, and the difference was statistically significant（LSD-t = 15.88,23.75;P = 0.01,0.00）. The effective clone numbers of cells in the FH and CT groups were（25.92 ± 5.22）and（364.00 ± 13.93） respectively. The number of effective clones in the FH group was significantly lower than that in the CT group（LSD-t =-37.67, P = 0.00）. Compared with the CT group, the number of G0/G1 cells in the FH group was significantly increased, the number of cells in the S phase and G2/M phase was significantly decreased. Conc