ATP50荧光表达载体构建及其在TM3小鼠睾丸Leydig细胞定位研究

Construction and Expression of ATP50 Fluorescent Protein in TM3 Mouse Leydig Cells

目的:构建ATP50的荧光表达载体,研究其在原代培养的小鼠睾丸Leyd ig细胞中的表达和在TM3小鼠睾丸Leyd ig细胞中的定位。方法:原代培养小鼠睾丸Leyd ig细胞并利用3β-HSD染色法鉴定,应用PCR方法研究ATP50在小鼠睾丸Leyd ig细胞中的表达。利用BamH I和EcoR I酶切位点把ATP50克隆到pEYFP-N1。细胞转染和细胞荧光显微镜技术观察YFP-ATP50在TM3小鼠睾丸Leydig细胞的定位。结果:ATP50表达在原代培养的小鼠睾丸Leydig细胞中。TM3小鼠睾丸Leydig细胞中,绿色荧光的YFP-ATP50和红色荧光的线粒体标记物M ito-tracker有完全的共定位。结论:ATP50在小鼠睾丸Leydig细胞表达,成功构建了ATP50真核荧光蛋白表达载体,明确YFP-ATP50定位在Leydig细胞的线粒体。这些结果将对老年男性睾丸间质细胞睾酮合成功能障碍的研究提供重要的信息,有利于进一步深入研究。

Objective： To study the expression and localization of ATP50 by construction of ATP50-pEYFP-N1 in primary cultured mouse Leydig cells. Methods： Primary cultured mouse Leydig cells were confirmed by 313-HSD staining. ATP50 was cloned into pEYFP-N1 between Barn HI and Eco RI sites. Cell-transfection and living-cell fluorescence imaging microscopy were employed to in- vestigate the sub-cellular localization of YFP-ATP50 in TM3 mouse Leydig cells. Results ： ATP50 green fluorescent protein was well co-localized with red fluorescence mitochondrion marker-Mitotracker in TM3 mouse Leydig cells. Conclusion ： ATP50 was expressed in primary cultured mouse Leydig cells. The fluorescent expression vector of ATP50 was constructed successfully and YFP-ATP50 was located in mitochondria in TM3 mouse Leydig cells, which provided a useful clue for further research on the steroidogenesis dysfunction in aging males.