SOX-9和胰岛素样生长因子1共同转染大鼠骨髓间充质干细胞向软骨细胞的分化

背景:单基因诱导虽能对骨髓间充质干细胞向软骨细胞分化产生积极影响,但作用有限,多种基因共同作用才符合机体真实内环境的需求,并有助于发挥多基因产物之间的协同作用,提高分化效果.目的:验证应用SOX-9和胰岛素样生长因子1基因转染大鼠骨髓间充质干细胞后,目的基因分泌情况及向软骨细胞的分化效果.设计、时间及地点:两样本观察,实验于2008-04/2009-02在中国医科大学细胞生物实验室完成.对象:6周龄健康雄性Wistar大鼠2只用于骨髓间充质干细胞提取.方法:扩增、提取质粒pcDNA3.1-IGF-1、pcDNA3.1-SOX-9.分离、纯化Wistar大鼠骨髓间充质干细胞.按转染情况分为5组:①未转染组:仅加入双无(无血清无双抗)L-DMEM.②转染窄载体组:双无L-DMEM+pcDNA3.1空载体和脂质体.③转染SOX-9组:双无L-DMEM+pcDNA3.1-SOX-9质粒和脂质体.④转染胰岛素样生长因子1组:双无L-DMEM分别+pcDNA3.1-IGF-1质粒和脂质体.⑤共转染组:双无L-DMEM分别+pcDNA3.1-IGF-1、pcDNA3.1-SOX-9质粒和脂质体,混合.主要观察指标:对转染后细胞进行筛选,MTT法测定筛选后细胞的增殖活性,反转录-聚合酶链反应和Western blot法检测筛选后细胞目的基因和蛋白的表达.结果:MTT法检测转染SOX-9组、转染胰岛素样生长因子1组和共转染组骨髓间充质干细胞增殖活性A值显著高于未转染组、转染空载体组(P<0.01).反转录-聚合酶链反应和Westem blot检测目结果显示,SOX-9表达以转染SOX-9组最多;胰岛素样生长因子1表达以共转染组最多:软骨细胞特异性基质Ⅱ型胶原表达以共转染组最多.结论:双基因转染在诱导骨髓间充质干细胞向软骨细胞转化的能力和细胞外基质分泌的能力上更明显高于单基质转染;另外结果间接说明胰岛素样生长因子1具有诱导骨髓间充质干细胞向软骨细胞分化的作用,但能力弱于SOX-9.

Differentiation of rat bone marrow mesenchymal stem cells to chondrocytes transfected by SOX-9 and insulin-like growth factor-1

BACKGROUND: Although single gene can induce bone marrow mesenchymal stem cells (BMSCs) into chondrocyte, it still has limit action, polygene interaction accord with real demand of body internal environment and is helpful to play synergistic effect and to improve the differentiated effect.OBJECTIVE: To investigate the secretion of target gene and differentiation of BMSCs transfected by BOX-9 and insulin-like growth factor-1 (IGF-1) gene alone and together into chondrocytes.DESIGN, TIME AND SETTING: Two-specimen observation was conducted in the Cell Biological Laboratory of China Medical University between April 2008 and February 2009. MATERIALS: Two 6-week-old healthy male Wistar rats were used for the BMSCs extraction. METHODS: The plasmids pcDNA3.1-1GF-1 and pcDNA3.1- BOX-9 were amplified and extracted. BMSCs of Wistar rats were separated and purified. According to the transfect situation, the BMSCs were divided into 5 groups. ①Non-transfected group:serum-free and double-antibody-free L-DMEM; ②empty vector transfection group: serum-free and double-antibody-free L-DMEM + pcDNA3.1 empty vector and liposome; ③BOX-9 transfection group: serum-free and double-antibody-free L-DMEM +pcDNA3.1-SOX-9 plasmid and liposome; ④IGF-1 transfection group: serum-free and double-antibody-free L-DMEM+pcDNA3.1-1GF-1 plasmid and liposome; ⑤SOX-9 and IGF-1 transfection group: serum-free and double-antibody-free L-DMEM+ pcDNA3.1-1GF-1 plasmid and liposome, pcDNA3.1-SOX-9 plasmid and liposome.MAIN OUTCOME MEASURES: After being transfected, the cells were selected, the proliferation activity was tested by MTT and expression levels of target gene and protein were tested by RT-PCR and Western blot. RESULTS: The proliferation activity (A value) of BMSCs transfected by SOX-9 alone, iGF-1 alone, SOX-9 and IGF-1 together was significantly higher than that of BMSCs in the non-transfected group and empty vector trensfection group (P < 0.01). RT-PCR and Western blot result showed that SOX-9 expression was the highest in the group transfected by SOX-9, the expression of IGF-1 was the highest in the group transfected both by SOX-9 and IGF-1, and the expression of collagen Ⅱ was the highest in the group transfected both by SOX-9 and IGF-1.CONCLUSION: The double gene transfection is supedor to single transfection with regard to the differentiation of BMSCs to chondrocytes and the secretion of extracallular matdx. It is indirectly suggested that IGF-1 induces BMSCs to differentiate into chondrocytes, but the capacity is less than SOX-9.