人转化生长因子-β1基因转染对人牙龈成纤维细胞成骨特性的影响

Influence of transfection with human transforming growth factor-β1 gene on the osteogenic potential of the cultured human gingival fibroblast

目的观察并评价基因转染后牙龈成纤维细胞(GF)表达外源目的基因人转化生长因子-β1(hTGF-β1)对其分化、成骨特性的影响。方法采用碱性磷酸酶(ALP)活性检测方法检测转染对GFALP活性的影响,免疫组化染色及图像分析评价基因转染后骨钙素(OC)、骨桥素(OPN)、骨涎蛋白(BSP)、骨结合素(ON)含量的变化,体外矿化结节形成实验检测转染后细胞成骨特性的变化。结果转染后GF的ALP活性较未转染组有显著的提高(P<0.05),并且与牙周膜成纤维细胞(PDLCs)的ALP活性接近(P>0.05)。OC含量检测显示转染后GF的OC含量与未转染组和PDLCs组相比,其差异均无统计学意义(P>0.05)。免疫组化染色后,转染组GF所表达的OPN、ON、BSP含量均高于未转染组GF(P<0.05),而与PDLCs间差异无统计学意义(P>0.05)。第21天和第24天时,在矿化液作用下PDLCs及转染GF在倒置显微镜下可见致密的结节形成,von Kossa染色可见紫色的矿化结节形成。未转染GF未见结节形成。而转染GF在未经矿化液诱导情况下,虽出现显著的细胞聚集,但von Kossa染色未见紫色矿化结节形成。结论转染pcDNA3-hTGF-β1后的GF可表达一定的成骨细胞特性,但这种成骨特性有限。

Objective To study the influence of transfection with human transforming growth factor-β1 （hTGF-β1） gene on the osteogenic potential and differentiation of the cultured human gingival fibroblast （GF）. Methods Enzyme kinetics method was used to measure the effects of the transfection on the alkaline phosphatase （ALP） activity. Immunohistochemistry stain and image analysis were applied to evaluate the alteration of the content of osteopontin （OPN）, bone sialoprotein （BSP）, osteonectin（ON）, osteocalcin （OC） in the GF with transfection. Mineralization nodules formation in vitro was also used. Results The ALP activity of the GF after transfection was higher than the GF without transfection significantly （P〈0.05）, and was close to that of the PDLCs （P〉0.05）. The content of OC in GF was not improved after transfection, was similar with that of PDLCs （P〉0.05）. Under immunohistochemistry stain, the contents of OPN, ON, BSP expressed in GF with transfection were higher than those of GF without transfection （P〈 0.05）, but similar to those of PDLCs （P〉0.05）. In the mineralized cultured medium, the nodules were observed in the GF with transfection and PDLCs after 21 days and 24 days alternatively. After von Kossa stain, purple mineralization nodules were observed. Conclusion The GF transfected with pcDNA3-hTGF-β1 could express some osteogenic characters, though these characters are restricted.