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本研究旨在探讨活化素(activin)对大鼠垂体GH3细胞中人生长激素(hGH)基因启动子活性的影响及其可能的调节机制。采用荧光素酶报告基因方法。首先建立含hGH基因启动子(-484～+30bp)和荧光素酶融合基因的稳定转染GH3细胞株,然后加入活化素或同时加入活化素与相关信号转导途径的激动剂,通过检测细胞培养液和细胞裂解液中GH的含量,以及GH3细胞内荧光素酶的变化,反映活化素对GH分泌、合成和hGH基因启动子活性的影响。将含不同长度hGH基因启动子序列的荧光素酶表达质粒分别转染GH3细胞,观察它们对活化素的反应,寻找活化素影响hGH基因启动子活性的关键DNA序列。结果表明,活化素(5,50nmol/L)能抑制大鼠垂体GH3细胞中GH的分泌和合成,活化素(5,50nmol/L)还能够抑制GH3细胞中hGH基因启动子的活性,使之仅达对照组的77%和69%;在胞内信号转导激动剂中,丝裂原活化蛋白激酶激酶(MAPKK/MEK)特异性激动剂C6ceramide(1μmol/L)完全取消了活化素对hGH基因启动子活性的抑制作用;活化素发挥抑制作用所需要的hGH基因启动子关键序列位于-132～-66bp之间。上述研究表明,活化素能抑制大鼠垂体GH3中hGH基因启动子的活性,它可能是通过抑制细胞内依赖MAPK的信号转导途径来完成的,同时hGH启动子上-132～-66bp的序列在其中发挥重要的作用。
The purpose of this study was to investigate the effect of activin on the promoter activity of human growth hormone (hGH) gene in pituitary cells and its possible regulatory mechanism. The luciferase reporter method was used. First, a stable transfected GH3 cell line containing the promoter of hGH gene (-484 ~ + 30bp) and luciferase fusion gene was established, then activin or activator and activator of related signal transduction pathway were added. The content of GH in culture solution and cell lysis solution and the change of luciferase in GH3 cells reflected the effects of activin on GH secretion, synthesis and hGH gene promoter activity. The luciferase expression plasmids containing different promoter sequences of hGH gene were transfected into GH3 cells respectively to observe their response to activin and to find out the key DNA sequences of activin affecting the promoter activity of hGH gene. The results showed that activin (5,50 nmol / L) could inhibit the secretion and synthesis of GH in pituitary GH3 cells. Activin (5,50 nmol / L) also inhibited the activity of hGH promoter in GH3 cells, Only 77% and 69% of the control group. In the intracellular signal transduction agonist, C6ceramide (1μmol / L), a specific agonist of mitogen-activated protein kinase kinase (MAPKK / MEK), completely abolished the effect of activin on hGH Gene promoter activity inhibition; activin required for the inhibition of the key hGH gene promoter sequence located between -132 ~ -66bp. The above study shows that activin can inhibit hGH gene promoter activity in rat pituitary GH3, which may be accomplished by inhibiting intracellular MAPK-dependent signal transduction, while the -132 ~ -66bp sequence on the hGH promoter In which play an important role.