课题背景：组织工程学及材料学的发展为软骨创伤生物学修复提供了极大可能，但软骨组织工程中种子细胞自身的缺陷使得这一技术的实际应用受到了极大限制，尤其是种子细胞复合培养或其向（类）软骨细胞定向分化过程中对外源性活性因子的依赖作用成为了相关研究的瓶颈，因而如何对各种种子细胞进行优化或改造成了人们的兴趣焦点。

创新要点：通过将胰岛素样生长因子1基因导入骨髓间充质干细胞使之自行表达所需的活性蛋白，有可能优化作为种子细胞的骨髓间充质干细胞的特性，使之更适于作为软骨组织工程的种子细胞。本实验发现胰岛素样生长因子1基因修饰后的骨髓间充质干细胞可以在一定时段内理想表达所需活性蛋白，为软骨组织工程提供了新的可供选择的种子细胞改良途径。

同行评价：实验通过构建携带功能基因胰岛素样生长因子1的真核载体并成功转染靶细胞，可以为组织工程的研究提供基因修饰的种子细胞，具有较重要的意义。实验选用了纳米载体，其安全性好，转染率高，目的基因在靶细胞有高效的表达，有较好的创新性。

摘要
目的: 构建增强型绿色荧光蛋白基因（enhanced-green fluorescent protein，EGFP）标记的人胰岛素样生长因子（human insulin-like growth factor, hIGF-1）真核表达载体，转染至兔骨髓间充质干细胞，为组织工程关节软骨的构建提供基因改良的种子细胞。
方法：实验于2005-03/2006-01在重庆医科大学儿科研究所完成。根据GeneBank公布的hIGF-1序列设计PCR引物，从质粒pcDNA3.1-hIGF-1中扩增出截短型hIGF-1，亚克隆至pMD-T18载体，测序鉴定后酶切出目的基因片段并插入pIRES2-EGFP中,构建成真核表达质粒pIRES2-EGFP-hIGF-1，经PCR及双酶切鉴定正确后用非病毒类纳米材料基因转移载体PAMAM-D介导转入兔骨髓间充质干细胞，通过荧光观察、流式细胞仪、RT-PCR等方法从各个水平检测hIGF-1在细胞的表达。
结果：成功构建了增强型绿色荧光蛋白基因标记的真核表达质粒pIRES2-EGFP-hIGF-1，并检测到目的基因hIGF-1在兔骨髓间充质干细胞的理想表达。
结论：新型纳米材料PAMAM-D是高效、简便、表达持久的基因转染方法，经IGF-1基因修饰的骨髓间充质干细胞是软骨组织工程种子细胞的理想选择。
关键词：纳米载体；人胰岛素样生长因子1；骨髓间充质干细胞；生物材料

丁幸坡，金先庆，王智勇，付艳丽. 纳米载体介导人胰岛素样生长因子1基因转染兔骨髓间充质干细胞及其表达[J].中国组织工程研究与临床康复，2008，12(6):1035-1038
[www.zglckf.com/zglckf/ejournal/upfiles/08-6/6k-1035(ps).pdf]

Transfection of human insulin-like growth factor-1 gene into rabbit mesenchymal stem cells mediated by nanometer vectors and the gene expression

Abstract

AIM：To gene-modify rabbit mesenchymal stem cells by transfection of eukaryotic vectors contained truncated human insulin-like growth factor-1 gene (hIGF-1) and enhanced-green fluorescent protein (EGFP) gene to provide gene-modified seed cells for tissue-engineered articular cartilage.
METHODS: The experiment was performed at Research Institute of Pediatrics, Chongqing Medical University from March 2005 to January 2006. After the amplification of truncated hIGF-1 gene from pcDNA3.1-hIGF-1 by polymerase chain reaction (PCR) according to GeneBank, the target gene fragment was inserted to plasmid pIRES2-EGFP to obtain recombinated vectors pIRES2-EGFP-hIGF1, which was identified with PCR and enzymatic digestion. Then, the transfection of restructured vectors to rabbit mesenchymal stem cells was performed mediated by nanometer vectors (polyamidoamine dendrimers, PAMAM-D). The expression of hIGF-1 gene was determined by inspection of fluorescence, flow cytometry, and RT-PCR.
RESULTS: pIRES2-EGFP-hIGF-1 labelled with enhanced-green fluorescent protein gene was constructed successfully and high efficient expression of hIGF-1 was detected in rabbit mesenchymal stem cells.
CONCLUSION: Gene transfection through nanometer vectors, PAMAM-D, is a simple, and highly effective method for gene expression in mesenchymal stem cells. The mesenchymal stem cell transfected with plasmid pIRES2-EGFP-hIGF1 is a good choice for seed cells of tissue-engineered articular cartilage.

Ding XP, Jin XQ, Wang ZY, Fu YL. Transfection of human insulin-like growth factor-1 gene into rabbit mesenchymal stem cells mediated by nanometer vectors and the gene expression.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(6):1035-1038(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-6/6k-1035(ps).pdf]