pcDNA3-hBMP2转染对成纤维细胞 生物学性状的影响

摘要

Objective To determine the effects of pcDNA3-hBMP2 transfectionon the profile of NIH3T3 fibroblasts. Methods pcDNA3-hBMP2 was constructed by using gene clone and recombined technique. With the help of lipofectamine, NIH3T3 fibroblasts were transfected with pcDNA3-hBMP2. The positive cell clones were selected with G418. The stable transfection and expression of hBMP2 in the NIH3T3 fibroblasts were determined by in situ hybridization and immunohistochemical analysis. The effect of pcDNA3-hBMP2 transfection on NIH3T3 cells proliferation was observed by MTT colorimetric assay. Flow cytometer was used to determine the effect of pcDNA3-hBMP2 transfection on cell cycle of NIH3T3.The ultrastructural changes of pcDNA3-hBMP2 transfected NIH3T3 cells were observed under transmission electron microscope. Alkaline phosphatase activity in both pcDNA3-hBMP2 transfected and non-transfected NIH3T3 cells was measured to observe the differentiation of fibroblasts into osteoblast lineage. Results pcDNA3-hBMP2 was successfully constructed. There was abundant BMP2 expression in NIH3T3 fibroblasts transfected with pcDNA3-hBMP2 confirmed by in situ hybridization and immunohistochemical analysis. There was no effect of pcDNA3-hBMP2 transfection on cell proliferation and cell cycle of NIH3T3.The ultrastructural features of pcDNA3-hBMP2 transfected NIH3T3 cells showed that there was massive rough endoplasmic reticulum in the cytoplasm, the cisternae were dilated to varying degrees and were full of abundant proteinoid substances. Alkaline phosphatase activity in pcDNA3-hBMP2 transfected NIH3T3 cells was much higher than that in non-transfected NIH3T3 cells. Conclusion There were no effects on cell proliferation and cell cycle of NIH3T3 by transfer of hBMP2 gene into NIH3T3 cells. The expression of hBMP2 in NIH3T3 cells could induce NIH3T3 cells differentiation into osteoblast lineage.%目的 探讨人BMP2基因转染对成纤维细胞NIH3T3生物学性状的影响。 方法 构建重组真核表达载体pcDNA3-hBMP2，并在脂质体介导下，将其导入NIH3T3成纤维细胞。通过G418筛选获得阳性克隆，用细胞原位杂交和免疫组织化学方法检测hBMP2基因在NIH3T3成纤维细胞内的表达情况；MTT法和FCM检测pcDNA3-hBMP2转染成纤维细胞后对细胞增殖和细胞周期的影响，透射电镜观察转染pcDNA3-hBMP2后成纤维细胞超微结构的改变，碱性磷酸酶的检测观察转染pcDNA3-hBMP2后成纤维细胞向成骨细胞分化情况。 结果 转染pcDNA3-hBMP2后的NIH3T3细胞内有大量hBMP2mRNA的转录及其蛋白的表达；转染pcDNA3-hBMP2对成纤维细胞增殖和细胞周期无影响；转染pcDNA3-hBMP2后的成纤维细胞超微结构可见粗面内质网丰富，囊腔扩张明显，其内充满中等电子密度的蛋白分泌物；碱性磷酸酶活性显著上升。 结论 pcDNA3-hBMP2转染对成纤维细胞NIH3T3增殖和细胞周期无影响，转染后的成纤维细胞不仅可形成BMP2，而且具有向成骨细胞系分化的特性。