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目的构建Cox7a2的荧光表达载体,研究其在小鼠睾丸Leydig细胞中的表达和定位。方法原代培养小鼠睾丸Leydig细胞并利用3β-HSD染色法鉴定,应用PCR方法研究Cox7a2在小鼠睾丸Leydig细胞中的表达。利用BglⅡ和EcoRⅠ酶切位点把Cox7a2克隆到pEYFP-N1。细胞转染和细胞荧光显微镜技术观察YFP-Cox7a2定位。结果Cox7a2表达在原代培养的小鼠睾丸Leydig细胞中。绿色荧光的YFP-Cox7a2和红色荧光的线粒体标记物- Mitotracker有完全的共定位。结论Cox7a2在小鼠睾丸Leydig细胞表达,成功构建Cox7a2真核荧光蛋白表达载体,明确YFP-Cox7a2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合成功能障碍的研究提供重要的信息。
Objective To construct a fluorescent expression vector for Cox7a2 and investigate its expression and localization in mouse Leydig cells. Methods Primary cultured Leydig cells of testes were identified by 3β-HSD staining. The expression of Cox7a2 in Leydig cells was detected by PCR. Cox7a2 was cloned into pEYFP-N1 using BglII and EcoRI restriction sites. YFP-Cox7a2 localization was observed by cell transfection and cell fluorescence microscopy. Results Cox7a2 was expressed in primary cultured mouse Leydig cells. Green fluorescent YFP-Cox7a2 and red fluorescent mitochondrial marker - Mitotracker complete co-localization. Conclusion Cox7a2 is expressed in Leydig cells of mouse testes. Cox7a2 eukaryotic expression vector was successfully constructed and YFP-Cox7a2 was localized in mitochondria of Leydig cells. These results will provide important information for the study of dysfunction of testicular interstitial cells in older men.