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

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。

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

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.