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目的前期研究发现呼吸链相关酶cox7A2和Atp合成酶亚单位Atp50在青年和老年睾丸组织中存在明显表达差异。本研究观察两者同源序列Atp50和coxⅦa在原代培养的大鼠Leydig细胞中的表达情况,为其在雄激素合成和睾丸衰退中的作用研究奠定基础。方法分离培养大鼠Leydig细胞,提取细胞总RNA,通过RT-PCR扩增目的片断,PCR产物进行TA克隆、测序鉴定。结果RT-PCR可见Atp50和coxVIIa表达,目的片段大小分别是438bp和358bp。PCR产物片段TA克隆测序结果与原序列一致率分别为99．7%和99．8%。结论RT-PCR和TA克隆测序结果证明大鼠睾丸Leydig细胞中存在coxVIIa和Atp50表达,对于其可能参与调节睾酮合成过程则有待于进一步研究。
Objective The previous study found that respiratory chain-related enzymes cox7A2 and Atp50 Atp50 subunit in the testicular tissue of young and old there is a significant difference. In this study, we observed the expression of homologous sequences Atp50 and coxⅦa in primary cultured rat Leydig cells and laid the foundation for their roles in androgen synthesis and testicular recession. Methods The rat Leydig cells were isolated and cultured, and the total RNA was extracted. The target fragment was amplified by RT-PCR. The PCR products were cloned by TA and sequenced. Results The expression of Atp50 and coxVIIa was observed by RT-PCR. The size of the target fragment was 438bp and 358bp, respectively. The coincidence rate of TA cloning and sequencing of the fragment of PCR product with the original sequence was 99.7% and 99.8% respectively. Conclusion The results of RT-PCR and TA cloning proved that there exists coxVIIa and Atp50 expression in Leydig cells of rat testes, and further study is needed for their possible involvement in the regulation of testosterone synthesis.