FGF-2真核表达质粒的构建及对C3H10细胞增殖及成软骨分化的影响

Construction of eukaryotic vector expressing FGF-2 gene and its effect on proliferation and cartilage differentiation of mesenchymal stem cells C3H10

目的:构建成纤维细胞生长因子-2(fibroblast growth factor-2,FGF-2)真核表达质粒,检测其对小鼠间充质干细胞C3H10增殖、成骨及成软骨分化作用的影响。方法:以质粒pAd-Trace-FGF-2为模板,PCR扩增目的基因FGF-2,经BglⅡ、SalⅠ双酶切后连接至pIRES2-EGFP表达载体中获得pIRES2-EGFP-FGF-2真核表达质粒,脂质体转染到C3H10细胞中,另设pIRES2-EGFP空载体转染对照组和空白细胞对照组。RT-PCR及Western blot法验证各组FGF-2的表达水平,MTT法和流式细胞术检测细胞的增殖活力及细胞周期分布,RT-PCR检测成骨及成软骨、Wnt信号通路相关基因的mRNA水平表达变化,Western blot法检测Ⅱ型胶原(collagenⅡ,ColⅡ)的表达,甲苯胺蓝染色检测软骨基质的表达。结果:重组表达质粒pIRES2-EGFP-FGF-2经双酶切及测序证实构建正确;质粒转染C3H10细胞后,其FGF-2基因的mRNA和蛋白水平表达较2个对照组明显增高,细胞增殖活力明显增高(P<0.05),软骨相关标志物Ⅰ型胶原(collagenⅠ,ColⅠ)、ColⅡ、蛋白聚糖(aggrecan,ACAN)mRNA转录水平较2个对照组均明显增高(P<0.05),骨钙蛋白(osteocalcin,OC)、骨保护素(osteoprotegerin,OPG)、骨桥蛋白(osteopontin,OPN)mRNA转录水平无明显升高(P>0.05),Wnt5a及Fzd8 mRNA转录水平降低(P<0.05),FGF-2转染组细胞甲苯胺蓝染色呈现紫红色异染。结论:成功构建FGF-2基因重组真核表达质粒,FGF-2过表达可提高C3H10细胞的增殖活力,对C3H10细胞有成软骨效应,但短期成骨效应不明显,可能与下调Wnt5a及受体Fzd8的表达有关。

Objective：To construct an eukaryotic vector of pIRES2-EGFP-FGF-2 and to investigate its effect on the proliferation, osteogenesis and ehondrogenesis of mouse mesenehymal stem ceils C3H10. Methods： FGF-2 gene was amplified by PCR from plasmid pAd-Traee-FGF-2. After being digested with BglⅡ and SalⅠ ,the PCR product was inserted into pIRES2-EGFP to construct pIRES2- EGFP-FGF-2. pIRES2-EGFP-FGF-2 plasmid was transfected into C3H10 by Liposomes, and C3H10 transfected with pIRES2-EGFP and untreated were set up as controls. The mRNA and protein expression levels of FGF-2 were determined by RT-PCR and Western blot respectively. The proliferation activity and cell cycle phase of C3H10 were examined by MTT and flow eytometry. The mRNA transcription levels of collagen Ⅰ （ Col Ⅰ）, osteocalcin （OC）, osteoprotegerin （OPG）, osteopontin （OPN）, collagen Ⅱ （Col Ⅱ）, aggrecan （ACAN） and Wnt pathway molecules were detected by RT-PCR. The protein expression of Col Ⅱ was detected by Western blot. Toluidine blue staining was carried out to measure the secretion of cartilage oligomeric matrix protein by cells in culture. Results： Re-striction analysis and sequencing proved that recombinant plasmid pIRES2-EGFP-FGF-2 was constructed correctly. Both the mRNA and protein expression level of FGF-2 increased signifi- cantly in plRES2-EGFP-FGF-2 transfected C3H10 cells. Cell proliferation activity of the experimental group increased significantly（P〈0.05）. The mRNA transcription levels of Col Ⅰ , ColⅡ and ACAN in C3H10 cells transfeeted with pIRES2-EGFP-FGF-2 recombinant plasmid increased significantly（P〈0.05）, however there was no significant increase in the mRNA expression of OC, OPG, OPN（P〉0.05）. The transcription levels of WntSa and Fzd8 decreased significantly（P〈0.05）. Toluidine blue staining showed purple metachromatic in the cells FGF-2 overexpressed C3HIO ceils. Conclusion ：The recombinant eukaryotic expression vector for FGF-2 gene is successfully constructed and overexpression of FGF-2 can promote chondrogenesis and proliferation activity of C3HIO cells but there is no significant effect on osteogenic differentiation in short-tenn. The effect of FGF-2 on chondrogenesis maybe involve in the downregulation of Wnt5a and Fzd8.