反转录病毒载体介导Indian hedgehog信号蛋白在C3H10T1/2细胞中的表达及成骨诱导潜能

摘要

BACKGROUND: Indian hedgehog (Ihh) has been confirmed to play an important role in regulating bone growth by a great amount of studies.OBJECTIVE: To clone the Ihh recombinant retrovirus vector, in addition, to observe its expression in C3H10T1/2 cells and study the effect on the osteogenesisMETHODS: According to the molecular biological technology, the Ihh gene sequence was linked into the retroviral vector pSFG vector which also contained the enhanced green fluorescence protein (EGFP) gene sequence. Viral supernatants harvested from package cell line were transfected into C3H10T1/2 cells. Then, the intensity of EGFP and the efficiency of gene transfer were monitored under the confocal microscope. The level of Ihh expression was detected by Western blot and morphologic changes of C3H10T1/2 cells were observed. Alkaline phosphatase staining was also used to determine the osteogenic differentiation.RESULTS AND CONCLUTION: The results of the confocal microscope and Western blot show that Ihh and EGFP proteins are coexpressed in a high level in C3H10T1/2 cells. The Ihh affects the morphology of C3H10T1/2 cell line significantly and induces the alkaline phosphatase expression in vitro. Therefore, C3H10T1/2 cells have the potential capability of differentiating into osteocytes.%背景:目前大量研究证实Indian hedgehog(Ihh)信号通路在骨发育中发挥着重要的调控作用.目的:克隆Ihh重组反转录病毒载体,观察Ihh在C3H10T1/2细胞中的表达及其成骨诱导效果.方法:采用分子克隆技术将Ihh基因与增强型绿色荧光蛋白基因克隆到反转录病毒载体pSFG上,包装出假病毒转染C3H10T1/2细胞,在共聚焦显微镜下观察其绿色荧光蛋白强度及转染效率.运用Western blot方法检测Ihh蛋白的表达,观察C3H10T1/2细胞形态学变化,碱性磷酸酶染色检测成骨活性.结果与结论:共聚焦显微镜下观察到大量的绿色荧光表达,Western blot检测结果显示Ihh基因及增强型绿色荧光蛋白基因在C3H10T1/2中呈显著共表达.在体外培养过程中,Ihh能诱导C3H10T1/2细胞形态学发生改变,增加碱性磷酸酶活性,提示Ihh具有诱导C3H10T1/2细胞向骨细胞分化的潜能.