中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (6): 934-939.doi: 10.3969/j.issn.2095-4344.2017.06.020

• 药物控释材料 drug delivery materials • 上一篇    下一篇

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

刘  云1,潘  杰2,3,董  伟1,2,惠  景1,2,李三华1,范  芳1,2,朱欣婷1,2 
  

  1. 1遵义医学院贵州省普通高等学校特色药物肿瘤防治特色重点实验室,贵州省遵义市  563000;2遵义医学院基础医学院,贵州省遵义市  563000;3仁怀市人民医院病理科,贵州省仁怀市   564500
  • 收稿日期:2017-01-13 出版日期:2017-02-28 发布日期:2017-02-28
  • 通讯作者: 朱欣婷,副教授,遵义医学院基础医学院,贵州省遵义市 563000
  • 作者简介:刘云,男,1980年生,贵州省遵义市人,汉族,2015年重庆大学毕业,博士,副教授,主要从事小分子抗癌药物的研究与开发研究。
  • 基金资助:

    贵州省科技厅社会发展攻关项目(黔科合SY[2013]3008);遵义医学院招标项目(F-614);贵州省教育厅特色重点实验室建设项目(黔教合KY字[2014]212)

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

Liu Yun1, Pan Jie2, 3, Dong Wei1, 2, Hui Jing1, 2, Li San-hua1, Fan Fang1, 2, Zhu Xin-ting1, 2
  

  1. 1Guizhou Provincial College-Based Key Lab for Tumor Prevention and Treatment with Distinctive Medicines, Zunyi Medical University, Zunyi 563000, Guizhou Province, China; 2Basic Medicine School of Zunyi Medical University, Zunyi 563000, Guizhou Province, China;3Department of Pathology, People’s Hospital of Renhuai, Renhuai 564500, Guizhou Province, China
  • Received:2017-01-13 Online:2017-02-28 Published:2017-02-28
  • Contact: Zhu Xin-ting, Associate professor, Guizhou Provincial College-Based Key Lab for Tumor Prevention and Treatment with Distinctive Medicines, Zunyi Medical University, Zunyi 563000, Guizhou Province, China; Basic Medicine School of Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • About author:Liu Yun, M.D., Associate professor, Guizhou Provincial College-Based Key Lab for Tumor Prevention and Treatment with Distinctive Medicines, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
  • Supported by:

    the Social Research and Development Project of Guizhou Science and Technology Department, No. SY[2013]3008; the Tender Project of Zunyi Medical University, No. F-614; the Key Lab Construction Project of the Educational Department of Guizhou Province, No. KY[2014]212

摘要:

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文题释义:
RNA干扰
:是基因沉默的常用方法,将小干扰 RNA安全高效地递送到肿瘤组织,使其在细胞内发挥基因沉默作用,是RNA干扰技术应用于肿瘤基因治疗的关键技术之一。目前,用于基因递送的载体主要有病毒载体和非病毒载体两种。病毒载体的优点是转染率高,但其存在安全隐患,在临床应用方面受到诸多限制;非病毒载体具有低毒、低免疫反应、易于组装等优点在基因递送方面应用前景广阔。
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。

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siRNA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siRNA-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、siRNA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siRNA-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、siRNA组、空白对照组(P < 0.05);④结果表明,LA-CS-PEI能实现siRNA的有效递送,并能沉默肝癌耐药细胞BEL7402/5-Fu中的MRE11基因。

关键词: 生物材料, 材料相容性, 半乳糖基化壳聚糖-聚乙烯亚胺, 去唾液酸糖蛋白受体, 减数分裂重组蛋白11, RNA干扰, 肝癌细胞BEL7402/5-Fu

Abstract:

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 (siRNA) 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/mL 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 siRNA-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 siRNA, respertively, the expressions of MRE11 mRNA 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/mL. The transfection efficiency was over 95% and the cell entry efficiency was extremely high when the mass ratio of GAL-CHI-g-PEI to siRNA-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 mRNA and protein in the composite transfection group were lower than those in the GAL-CHI-g-PEI, siRNA and control groups (P < 0.05). These results suggest that the effective delivery of siRNA and the silencing of the MRE11 gene in BEL7402/5-Fu cells can be achieved by GAL-CHI-g-PEI.

Key words: Chitosan, Transfection, Gene Silencing, Polyethyleneimine, Tissue Engineering

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