摘要
目的扩增FasL启动子并构建带有FasL启动子的荧光素酶报告质粒,并评价在皮质酮(啮齿类动物体内的糖皮质激素)作用的睾丸Leydig细胞中,由FasL启动子调控的荧光素酶的表达活性。本研究将为进一步分析Leydig细胞中FasL启动子转录调控的作用机制奠定基础。方法采用DNA重组技术构建由FasL启动子调控的荧光素酶报告基因的表达质粒。此重组质粒经阳离子脂质体LipofectAMINE介导,瞬时转染入Leydig细胞中,36h后细胞经皮质酮处理,并于48h时收集细胞。测定荧光素酶的相对表达活性。结果(1)酶切及测序证实成功扩增FasL基因启动子并构建了带有FasL基因启动子的报告基因表达质粒；(2)皮质酮处理的Leydig细胞中FasL启动子调控的荧光素酶表达质粒的表达活性显著增高。结论构建成功的带有FasL启动子的荧光素酶报告质粒可准确地评价FasL启动子在凋亡过程中的作用。
Objectives To investigate how the FasL expression is triggered in response to CORT in Leydig cells. Methods Luciferase expression vectors with FasL gene promoter- pGL3-FLP689-1uc （-618- ＋71 ） was constructed. This vector was transfected into Leydig cells using the LipofectamineTM 2000 reagent. Thirty-six hours following transfection, the cells were treated with corticosterone （CORT） for 12h. At 48 h after transfection, the activities of firefly and Renilla luciferases were measured with the Dual-Luciferase Reporter assay system, and expressed as relative light units. Results pGL3-FLP689-1uc was successfully constructed and the activity of the FasL promoter at -618- ＋71 could be remarkably enhanced after the Leydig cells were subjected to CORT treatment. Condusion The result suggests that CORT treatment enhanced FasL transcriptional activity in Leydig cells. The data show that the -618- ＋71 region is sufficient for CORT-mediated activation of FasL expression and served as a basis for mutational analysis.
出处
《中国男科学杂志》
CAS
CSCD
2006年第4期4-6,10,共4页
Chinese Journal of Andrology
基金
国家自然科学基金(30570681)
