中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (38): 7095-7099.doi: 10.3969/j.issn.1673-8225.2010.38.016

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

新型阳离子脂质体制备及其体外转基因特征

蒋福升，王辉平，刘  霞，邱华飞，孙  敏，张  英，张伟威，丁志山   

  1. 浙江中医药大学，浙江省杭州市  310053
  • 出版日期:2010-09-17 发布日期:2010-09-17
  • 通讯作者: 丁志山，教授，博士，硕士生导师，浙江中医药大学，浙江省杭州市 310053 zjtcmdzs@163.com
  • 作者简介:蒋福升★，男，1982年生，浙江省兰溪市人，汉族，浙江中医药大学在读硕士，主要从事中西医结合基础方面的研究。 Jfs1020@163.com
  • 基金资助:

    浙江省科技厅新苗计划基金项目(2008R40G2120030)。

New cationic liposome preparation and transgenic evaluation in vitro

Jiang Fu-sheng, Wang Hui-ping, Liu Xia, Qiu Hua-fei, Sun Min, Zhang Ying, Zhang Wei-wei, Ding Zhi-shan   

  1. Zhejiang Chinese Medical University, Hangzhou   310053, Zhejiang Province, China
  • Online:2010-09-17 Published:2010-09-17
  • Contact: Ding Zhi-shan, Professor, Doctor, Master’s supervisor, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China zjtcmdzs@163.com
  • About author:Jiang Fu-sheng★, Studying for master’s degree, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China Jfs1020@163.com
  • Supported by:

    Xinmiao Plan of Zhejiang Provincial Science and Technology Ministry, No. 2008R40G2120030*

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299

被引次数

2

摘要：

背景：阳离子脂质体及胶束、纳米粒等转基因载体是目前非病毒转基因载体领域研究热点；而目前市售转基因试剂盒价格昂贵，并且基本上都是进口商品，如果能够开发一种价廉，转基因效率高，具有自主知识产权的转基因试剂盒将展现广阔市场前景。
目的：合成一种新型阳离子脂质复合物，并制备相应阳离子脂质体，对其体外转基因效率进行考察。
方法：以1-乙基-3- (3-二甲胺丙基)碳二亚胺、胆固醇、丁二酸酐为原料合成阳离子脂质复合物，采用薄层色谱、红外光谱等方法对产物进行结构表征。然后用薄膜分散法，将适当比例的二油酰磷脂酰乙醇胺和阳离子脂质复合物混合制备成阳离子脂质体，并用其包封含绿色荧光蛋白报告基因的真核表达质粒pEGFPC1，对人宫颈癌Hela细胞株进行体外转染。
结果与结论：通过两步法合成了一种全新的阳离子脂质复合物——胆固醇-丁二酸酯-1-乙基-3- (3-二甲胺丙基)碳二亚胺，薄层色谱及红外光谱结果表明合成了预期产物。薄膜分散法制得阳离子脂质体，其中以这种阳离子脂质复合物和二油酰磷脂酰乙醇胺摩尔比为1∶1左右所制备的脂质体颗粒较为均匀、细小，形态基本呈圆形。体外对质粒包裹实验结果表明，阳离子脂质体与质粒比为5∶3，可完全包封质粒。用该阳离子脂质体对Hela细胞进行转染，24 h后，瞬时转染效率达35.6%。从初步实验结果看，所合成的阳离子脂质复合物结构总体与市售阳性成分结构类似，合成方法简单、廉价，体外对质粒DNA有很好包裹性能，细胞毒性低，然而体外对Hela细胞的转染效率还明显偏低。

关键词: 胆固醇, 1-乙基-3- (3-二甲胺丙基)碳二亚胺, 阳离子脂质体, 质粒DNA, 绿色荧光蛋白

Abstract:

BACKGROUND: Cationic liposomes and transgenic vectors such as micelles and nanoparticles, is a current research focus of non-viral transgenic vector; currently commercial transgenic kits are expensive and basically imported goods, it is increasing urgent to develop a cheap, efficiently transgenic kit with independent intellectual property rights for broad market prospects.
OBJECTIVE: To synthesize a new cationic lipid composite and prepare cationic liposome, to investigate its transgenic efficiency in vitro.
METHODS: The cationic lipid composite was synthesized by organic chemistry synthesis method using EDCI, cholesterol, succinic anhydride as raw materials; the structure characterization of the products was validated through thin-layer chromatography and infrared spectrum technique. A new cationic liposome was prepared with proper proportion of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine and cationic lipid composite, and was used to envelop eukaryotic expression plasmid pEGFPC1 containing green fluorescent protein report gene, and then its gene transfection efficiency on human cervical carcinoma Hela cell in vitro was detected.
RESULTS AND CONCLUSION: A new cationic lipid composite, Cholesterol-Succinate-EDCI, was obtained with two step methods; thin-layer chromatography and infrared spectrum confirmed it as the expect product. The liposome were prepared by film dispersion method with 1:1 molar ratio of Cholesterol-Succinate-EDCI: 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, the liposome granules were tiny, spherical shaped and well-distributed. In vitro plasmid enveloping experiment showed that, this liposome can completely envelop plasmid and form lipoplex with the mass ratio 5:3. In vitro gene transfer essay indicated that the lipoplex transient transfection efficiency can reach 35.6% after treatment with 24 hours. The prepared cationic lipid composite structure is similar to commercially positive component structure, the synthesis method is simple and cheap, it can effectively envelop the plasmid DNA in vitro, with low toxicity, however, in vitro transfection efficacy of Hela cells remains low.

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