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目的:探讨细胞因子白介素-11(IL-11)、睫状神经营养因子(CNTF)和转化生长因子(TGF-β)对大鼠垂体MtT/S细胞中人生长激素(hGH)基因启动子活性的影响及其与垂体特异性转录因子Pit-1蛋白的关系。方法:采用荧光素酶报告基因的方法。首先建立含hGH基因启动子(-484～30 bp)和荧光素酶融合基因的稳定转化MtT/S细胞系,然后用细胞因子刺激,检测细胞培养液和细胞裂解液中GH的含量,反映它们对GH分泌和合成的影响;检测MtT/S细胞内荧光素酶的变化,说明细胞因子对hGH基因启动子活性的作用。将Pit-1蛋白表达质粒(pcDNA-pit-1-cDNA)单独转染或与Pit-1反义寡核苷酸(Pit-1OND)共转染于稳定转化的MtT/S细胞中,观察加入细胞因子后荧光素酶的变化,探讨细胞因子的作用与Pit-1蛋白的关系。结果:IL-11(20 nmol/L)、CNTF(10 nmol/L)能刺激大鼠垂体MtT/S细胞中GH的分泌和合成,增强MtT/S细胞中荧光素酶的表达,分别增加到对照组的134%、122%。TGF-β(5 nmol/L)能减少GH的分泌和合成,抑制荧光素酶的表达到对照组的72%。Pit-1蛋白过表达和表达被抑制对细胞因子的调节作用没有影响。结论:IL-11、CNTF和TGF-β通过调节大鼠垂体MtT/S细胞中hGH基因启动子活性影响GH的合成,Pit-1蛋白可能不参与这一调节作用。
Objective: To investigate the effects of cytokines interleukin-11 (IL-11), ciliary neurotrophic factor (CNTF) and transforming growth factor-β (TGF-β) on the promoter activity of human growth hormone (hGH) gene in rat pituitary And its relationship with pituitary-specific transcription factor Pit-1 protein. Methods: Methods using luciferase reporter gene. First, a stable transformed MtT / S cell line containing the promoter of hGH gene (-484 ~ 30 bp) and luciferase fusion gene was constructed and then stimulated with cytokines to detect the content of GH in the cell culture solution and cell lysate, reflecting their On GH secretion and synthesis; detection of MtT / S intracellular luciferase changes, cytokines on hGH gene promoter activity. The Pit-1 protein expression plasmid (pcDNA-pit-1-cDNA) was transfected either alone or with Pit-1 antisense oligonucleotides (Pit-1OND) in stably transformed MtT / S cells, Cytokines luciferase changes to explore the role of cytokines and Pit-1 protein. Results: IL-11 (20 nmol / L) and CNTF (10 nmol / L) stimulated the secretion and synthesis of GH in the pituitary of MtT / S cells and increased the expression of luciferase in MtT / S cells The control group 134%, 122%. TGF-β (5 nmol / L) reduced the secretion and synthesis of GH, and inhibited the expression of luciferase to 72% of the control group. Inhibition of Pit-1 protein overexpression and expression has no effect on the regulation of cytokines. Conclusion: IL-11, CNTF and TGF-β may affect the synthesis of GH by regulating the promoter activity of hGH gene in rat pituitary. Pit-1 protein may not participate in this regulation.