中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (16): 2920-2924.doi: 10.3969/j.issn.1673-8225.2011.16.016

• 纳米生物材料 nanobiomaterials • 上一篇    下一篇

mPEG-CS纳米粒介导livin shRNA基因纳米复合物的制备及转染效率

肖  维，彭  健，张阳德   

  1. 中南大学卫生部肝胆肠外科研究中心,湖南省长沙市 410008
  • 收稿日期:2011-01-12 修回日期:2011-03-14 出版日期:2011-04-16 发布日期:2013-11-11
  • 通讯作者: 张阳德，博士，博士生导师，教授，中南大学卫生部肝胆肠外科研究中心，湖南省长沙市 410008
  • 作者简介:肖维★，男，1984年生，湖南省南县人，中南大学在读硕士，医师，主要从事纳米载体介导的凋亡诱导基因治疗肿瘤的临床前研究。 Hahashu2005@yahoo.com.cn
  • 基金资助:

    国家“863”计划重点项目，课题名称：非病毒载体介导的凋亡诱导基因治疗肿瘤的临床前研究。

Preparation and transfection efficiency of mPEG-CS/livin shRNA complex

Xiao Wei, Peng Jian, Zhang Yang-de   

  1. National Hepatobiliary ＆ Enteric Surgery Research Center of Central South University, Changsha  410008, Hunan Province, China
  • Received:2011-01-12 Revised:2011-03-14 Online:2011-04-16 Published:2013-11-11
  • Contact: Zhang Yang-de, Doctor, Doctoral supervisor, Professor, National Hepatobiliary ＆ Enteric Surgery Research Center of Central South University, Changsha 410008, Hunan Province, China
  • About author:Xiao Wei★, Studying for master’s degree, Physician, National Hepatobiliary ＆ Enteric Surgery Research Center of Central South University, Changsha 410008, Hunan Province, China Hahashu2005@yahoo.com.cn
  • Supported by:

    the National 863 Program of China, No. 2007AA021009*

PDF

398

被引次数

9

摘要：

背景：作为非病毒基因转染载体，由可降解的高聚物形成的纳米载体目前被广泛由于基因转染，因为他们具有良好的缓释性，靶向性和生物相容性。
目的：制备mPEG-CS纳米粒，探讨mPEG-CS作为Livin shRNA基因转染载体的可行性。
方法：通过离子交联法制备mPEG-CS纳米粒，利用聚乙二醇对壳聚糖进行改性，通过静电吸附法制备载livin shRNA的基因纳米复合物。Zeta-size分析仪和透射电镜检测空白纳米粒和载livin shRNA的基因纳米复合物的形态、粒径和zeta电位，测定基因纳米复合物的包封率，凝胶电泳阻滞实验和DNase I酶消化实验验证纳米粒对基因的保护作用。利用最佳条件下制备的基因纳米复合物，转染大肠癌HT-29细胞，考察转染效率。
结果及结论：成功制备出约60 nm的mPEG-CS纳米粒，当纳米粒与基因体积比为3∶1时，得到的基因纳米复合物形较规则，粒径100 nm左右；其包封率为(94.32±0.35)%。凝胶电泳阻滞实验表明纳米粒能够紧密结合DNA，对基因具有良好的基因保护作用。该基因纳米复合物转染大肠癌细胞的转染效率高,持续作用时间长。mPEG-CS纳米粒作为基因转染载体，对基因具有保护作用，能够将livin shRNA重组质粒高效转染入大肠癌细胞，能够在大肠癌细胞内长时间表达，克服了RNA干扰在基因治疗肿瘤中基因作用时间较短的缺点。

关键词: mPEG-CS纳米粒, Livin shRNA, 大肠癌, 转染效率, 基因

Abstract:

BACKGROUND: Nanoparticles formulated from biodegradable polymers such as mono methyl poly (ethylene glycol)-g-chitosan are being extensively investigated as non-viral gene delivery systems due to their sustained release characteristics, targeting and biocompatibility.
OBJECTIVE: To prepare mPEG-CS nanoparticles and to explore the feasibility of mPEG-CS as livin shRNA gene carrier, and to study the gene transfection efficiency to colorectal cancer cell HT-29 mediated mPEG-CS nanoparticles.
METHODS: mPEG-CS nanoparticles were prepared by ionic cross-linking method and mPEGylated -chitosan/ livin shRNA complex by Static adsorption. The pattern, size and Zeta potential of blank nanoparticles and mPEGylated-chitosan/ livin shRNA complex were detected by Zeta-size analyzer and transmission electron microscope (TEM). The encapsulation efficiency of gene-nano complex was measured. The protection of nanoparticles for gene was validated through Gel electrophoresis block experiment and DNase I enzyme digestion experiment. The transfection efficiency of livin shRNA was compared to that of CRC cell HT-29 mediated by mPEG-CS nanoparticles or not.
RESULTS AND CONCLUSION: mPEG-CS nanoparticles of the size about 60 nm were successfully prepared out, when nanoparticles /gene volume ratio was 3∶1, size of gene-nano complex was 100 nm and encapsulation efficiency was (94.32±0.35)%. The gel electrophoresis blocking test showed that nanoparticles could be effectively combined with the plasmid, and Dnase I test proved that the nanoparticles could protect the plasmid. Transfection efficiency mediated by mPEG-CS was higher than naked gene and working a longer time. As gene carrier, mPEG-CS nanoparticles can protective gene well, and it is able to transfect livin shRNA recombinant plasmid into colorectal cancer cells and express in a long time. The gene-nano complex overcomes the shortage of relatively short time of RNA interference in gene therapy for tumor.

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