半乳糖基化壳聚糖-聚乙烯亚胺递送siRNA对肝癌耐药细胞BEL7402/5-Fu中MRE11表达的影响

Effect of small interfering RNA delivery using galactosylated chitosan-graft-polyethylenimine on MRE11 gene expression in drug-resistant hepatocellular carcinoma BEL7402/5-Fu cells

背景:壳聚糖是一种常用的非病毒载体,具有良好生物相容性、生物可降解性、低毒等优点,但其基因递送能力相对较弱,常需要进行结构衍生修饰。目的:观察半乳糖基化壳聚糖-聚乙烯亚胺(LA-CS-PEI)在肝癌耐药细胞BEL7402/5-Fu中递送si RNA的能力及对减数分裂重组蛋白11(MRE11)表达的影响。方法:1将肝癌耐药细胞BEL7402/5-Fu或人肝细胞HL-7702分别与0,10,20,40,80,100 mg/L的LA-CS-PEI或壳聚糖-聚乙烯亚胺共培养24 h,检测细胞存活率;2将LA-CS-PEI与si RNA-FAM分别以0,2,4,8,16,32的质量比混合,制备复合转染颗粒。以复合转染颗粒转染肝癌耐药细胞BEL7402/5-Fu,48,72,96 h后,以转染效率筛选最佳质量比与转染时间,进行下步实验;3以复合转染颗粒、LA-CS-PEI、si RNA分别转染肝癌耐药细胞BEL7402/5-Fu 48 h后,以未转染的细胞为空白对照,检测MRE11基因及蛋白的表达。结果与结论:1与壳聚糖-聚乙烯亚胺相比,LA-CS-PEI毒性更低,且当LA-CS-PEI质量浓度高达100 mg/L时,仍表现出很低的细胞毒性;2LA-CS-PEI与si RNA-FAM质量比为4和8时转染效率均达95%以上,入胞效率极高,转染48 h转染效果最好,所以后续实验选择质量比为8,转染时间为48 h的复合转染颗粒;3复合转染颗粒组MRE11基因及蛋白表达低于LA-CS-PEI组、si RNA组、空白对照组(P<0.05);4结果表明,LA-CS-PEI能实现si RNA的有效递送,并能沉默肝癌耐药细胞BEL7402/5-Fu中的MRE11基因。

BACKGROUND： Chitosan is a kind of common non-viral vector characterized by good biocompatibility, biodegradability and low toxicity. However, it usually needs structural modification via derivatization due to its weak gene delivery ability. OBJECTIVE： To observe the ability of galactosylated chitosan-graft-polyethylenimine（GAL-CHI-g-PEI） delivering small interfering RNA（si RNA） on drug-resistant hepatocellular carcinoma BEL7402/5-Fu and its effect on the expression of meiotic recombination 11（MRE11）.METHODS： BEL7402/5-Fu cells or human hepatocytes HL-7702 were co-cultured with 0, 10, 20, 40, 80 and 100 μg/m L GAL-CHI-g-PEI or chitosan-PEI for 24 hours, and then the cell viability was detected. The composite transfection particles were prepared by mixing GAL-CHI-g-PEI with si RNA-FAM at the mass ratio of 0, 2, 4, 8, 16 and 32, respectively. The next experiments were performed based on BEL7402/5-Fu cells transfected with the compound transfection particles, and the optimal mass ratio and transfection time were selected according to the efficiency of transfection at 18, 72 and 96 hours afte transfection. At 48 hours after BEL7402/5-Fu cells transfected with compound transfection particles, GAL-CHI-g-PEI and si RNA, respertively, the expressions of MRE11 m RNA and protein were detected in comparison with untransfected cells as controls. RESULTS AND CONCLUSION： Compared with chitosan-PEI, GAL-CHI-g-PEI demonstrated lower toxicity, and still showed low cytotoxicity when the mass concentration reached 100 μg/m L. The transfection efficiency was over 95% and the cell entry efficiency was extremely high when the mass ratio of GAL-CHI-g-PEI to si RNA-FAM was 4 and 8, respectively, with the optimal transfection effect at 48 hours after transfection. Therefore, the mass ratio in the subsequent experiments was set to 8. The expressions of MRE11 m RNA and protein in the composite transfection group were lower than those in the GAL-CHI-g-PEI, si RNA and control groups（P〈0.05）. These results suggest that the effective delivery of si RNA and the silencing of the MRE11 gene in BEL7402/5-Fu cells can be achieved by GAL-CHI-g-PEI.