成纤维细胞生长因子诱导骨髓间充质干细胞分化腱细胞的研究

Fibroblast growth factor 2 induces bone marrow stem cells to differentiate into tenocytes

目的检测腺病毒携带成纤维细胞生长因子2(FGF2)转染骨髓间充质干细胞(BMSCs)后FGF2的表达情况并探索其对细胞增殖、分化及韧带相关特异性细胞外基质表达的影响。方法将腺病毒进行细胞转染,转染后将细胞分为空白组(正常培养)、对照组[重组腺病毒(Ad.e GFP)转染]和试验组[空病毒(Ad.b FGF2-e GFP)转染]。采用形态学、流式细胞仪观察、实时荧光定量聚合酶链反应(q RT-PCR)、蛋白质印迹法(WB)检测转染后BMSCs中FGF2的表达。并采用MTT法检测过表达FGF2后BMSCs增殖的变化。最后用q RT-PCR及WB检测BMSCs中带韧带相关特异性细胞外基质基因的表达水平。结果试验组转染后FGF2表达最高,与空白组及对照组相比,差异有统计学意义(t=19.648,P<0.001;t=17.506,P<0.001);MTT结果示:在转染后第3天,与空白组及对照组相比,试验组可明显促进BMSCs的增殖,差异有统计学意义(t=3.401,P=0.015;t=3.234,P=0.018);过表达FGF2基因可明显上调BMSCs中特异性细胞外基质基因Ⅰ型胶原蛋白(ColⅠ)、Ⅲ型胶原蛋白(ColⅢ)、纤维连接蛋白(FN)、波形蛋白(Vimentin)及α平滑肌肌动蛋白(α-SMA)的表达。结论 FGF2能促进BMSCs的增殖并向腱细胞分化,腺病毒携带FGF2转染BMSCs有望成为组织工程韧带的种子细胞。

Objective To detect the expression of fibroblast growth factor 2（FGF2） in human bone marrow stem cells（BMSCs）transfected with the Ad.FGF2-e GFP and the effect of FGF2 on proliferation, differentiation, and production of specific extracellular matrix proteins of BMSCs. Methods The BMSCs were divided into three groups： blank group, Ad.e GFP group and Ad.b FGF2-e GFP group. The expression of FGF2 in BMSCs was detected by general morphological characteristics, flow cytometry, real-time quantitative polymerase chain reaction（q RT-PCR）, and Western bloting（WB）. The effect of FGF2 overexpression on BMSCs proliferation was assessed by MTT assay. q RT-PCR and WB were performed to detect the expression of ligament specific extracellular matrix gene in BMSCs treated with Ad.FGF2-e GFP. Results FGF2 was overexpressed in BMSCs treated with Ad.FGF2-e GFP, compared to blank group or Ad.e GFP group, there were significantly statistical differences（t =19.648,P 0.001; t =17.506,P 0.001）. Overexpression of FGF2 significantly promoted BMSCs proliferation after 3 days treatment（blank group： t =3.401,P =0.015; Ad.e GFP group： t =3.234,P =0.018）. Notably,overexpressing of FGF2 gene up-regulated the Collagen Ⅰ, Collagen Ⅲ, Fibronectin, Vimentin, and α-SMA. Conclusion FGF2 promotes BMSCs proliferation and differentiates into tenocytes. BMSCs transfected with the Ad.FGF2-e GFP can be the seed cells for tissue-engineered ligament.