Abstract

BACKGROUND:Transfection is the most important beginning component of research about gene function. It is a problem to find a transfection agent with high efficiency and safety. Nanosized materials have high surface activity, are easy to be modified and easier to pass the biomembrane. Researchers are studying how to use nanosized materials as a transfection agent.
OBJECTIVE: To study the transfection efficiency of different basic polymers with different molecular mass and degree of substitute, and to find the optimized transfection agent.
DESIGN: Controlled study.
SETTING: Institute of Genetic Engineering, Southern Medical University.
METHODS: This study was performed in the Institute of Genetic Engineering, Southern Medical University from March 2006 to June 2007. Using the lipofectamine reagent as the positive control, we transfected siRNA (0.2 nmol/L), FITC-labeling targeting bcl-2, by nine nanometers (polylactic acid-polyglycolic acid, chitosan-poly-caprolactone, polyethyleneimin-macrogol) into leukemic cell K562 cultured without serum. Six hours post-transfection, 20% FBS serum was added. The cell proliferation was measured at 24, 48 and 72 hours by using the MTT method. After transfecting for 48 hours, the cells were collected to detect transfection ratio by using fluorescent microscopy, apoptosis ratio and expression of K562 and bcl-2 protein by flow cytometer (FCM).
RESULTS: ① Fluorescence microscope detection showed there were significant differences of transfection efficiency between different materials (P < 0.05). Moreover there were statistical differences between different degrees of substitute, although they were the same material (P < 0.05). ② MTT method indicated the cell proliferation ratio was positively related to transfection ratio. ③ Flow cytometry results showed the suppression of expression of targeting gene and apoptosis ratio were positive correlated with transfection ratio.
CONCLUSION: The nanometer poly-ethylene glycol combined with poly-ethylene imine (PEG-PEI), whose molecular mass is 1 800/2 000 and degree of substitute is 29%, has a high efficiency and low toxicity.

Institute of Genetic Engineering, South-ern Medical Univer-sity, Guangzhou 510515, Guangdong Province, China

Hu Hai-yan☆, Doctor, Attending physician, Institute of Genetic Engineering, Southern Medical University, Guang-zhou 510515, Guangdong Province, China
xqgs1104@yahoo.
com.cn

Correspondence to: Ma Wen-li, Institute of Genetic Engineer-ing, Southern Medi-cal University, Guangzhou 510515, Guangdong Province, China

Supported by: a grant from the Health Department of Guangdong Province, No. WSTJJ20061116510106197611042927*

Received: 2007-12-28 Accepted: 2008-01-18 (07-50-12-7271 / N)

Hu HY, Zhang MX, Guan XP, Jiang H, Ma WL.Optimizing siRNA transfection by nanoparticle cationic poly-mer.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(6): 1145-1148(China)
[www.zglckf.com/zglckf/ejournal/upfiles/ 12-6/6k-1145
(ps).pdf]

摘要
背景：转染是基因研究最关键的始动环节，高效安全的基因转染试剂是基因研究的热点问题。纳米级材料表面活性强，易于表面修饰，膜通透性高，如何将物质纳米化应用于基因转染尚在探索之中。
目的：观察不同分子质量、结脂度的纳米级阳离子聚合物的转染效率，筛选低毒、高效的优化转染试剂。
设计：对照实验。
单位：南方医科大学基因研究所。
方法：实验于2006-03/2007-06在南方医科大学基因工程研究所实验室完成。以脂质体为阳性对照，将9种不同分子质量、结脂度的纳米级阳离子聚合物聚乳酸聚乙醇酸、壳聚糖-聚己内酯、聚乙烯亚胺-聚乙二醇，包裹FITC标记的以bcl-2基因为靶标的siRNA（0.2 nmol/L），转入无血清培养的白血病细胞株K562，于转染后6 h后补加20%胎牛血清培养基。MTT法测定24，48，72 h后细胞增殖状况，荧光显微镜检测转染48 h后各纳米材料转染效率，流式细胞仪检测白血病细胞株K562细胞Bcl-2蛋白的表达和凋亡率。
结果：①荧光显微镜结果:不同材料的纳米级颗粒，其转染效率有统计学差异（P < 0.05），同种材料的纳米结构因其结脂度不同，其转染效率也有统计学差异（P < 0.05）。②MTT结果：表明细胞增殖率与转染率正相关。③流式细胞仪结果表明:转染siRNA引起的靶基因表达抑制以及细胞凋亡率与转染效率正相关。
结论：分子质量为1 800/2 000，脂结合力为29%的纳米级聚乙烯亚胺-聚乙二醇颗粒，为低毒、高效的转染试剂。
关键词：Bcl-2；siRNA；纳米；生物材料

胡海燕，张梅霞，官习鹏，姜 铧，马文丽
南方医科大学基因研究所，广东省广州市 510151
胡海燕☆，女，1976年生，河南省南阳市人，汉族，南方医科大学基因芯片研究所博士后，主治医师，主要从事肿瘤基因治疗和干细胞移植研究。
通讯作者：马文丽，博士后导师，南方医科大学基因芯片研究所，广东省广州市 510151
广东省卫生厅基金资助项目（WSTJJ20061116510106197611042927）*

中图分类号: R318.08 文献标识码: A 文章编号: 1673-8225(2008)06-01145-04
胡海燕，张梅霞，官习鹏，姜铧，马文丽.纳米级阳离子聚合物优化转染siRNA的研究[J].中国组织工程研究与临床康复，2008，12(6):1145-1148
[www.zglckf.com/zglckf/ejournal/upfiles/08-6/6k-1145(ps).pdf]
(Edited by Brain Wilson/Ji H/Wang L)