B淋巴细胞中生长激素基因转录调控机制的研究

【作者】 栾好江；

【导师】 邓洁英； 史轶蘩；

【作者基本信息】 中国协和医科大学 ， 内科学， 2001， 博士

【摘要】 在激素与免疫系统的相互关系中，由于GH有增强免疫功能的作用，它与免疫系统的关系一直被学者们所关注。人和啮齿类动物的淋巴细胞中都能够表达GH及其受体。故淋巴细胞分泌的GH有可能以自分泌和旁分泌的方式影响淋巴细胞的功能。目前，淋巴细胞中GH表达的调节机制还不清楚。国外的学者们从细胞水平观察到一些激素能影响淋巴细胞的GH分泌。此外，细胞因子可以调节淋巴细胞的生长、分化和功能。但是激素和细胞因子对淋巴细胞中GH表达是否有调节作用呢?我们用荧光素酶报告基因的方法，探讨了这个问题。此外，我们还研究了B淋巴细胞中胞内信息传递途径与GH基因转录的关系；不同长度GH基因启动子的活性以及垂体特异性转录因子Pit-1与B淋巴细胞中GH基因转录的关系等问题。 1．激素和细胞因子对B淋巴细胞中GH基因转录的影响：首先把人GH基因调控序列(-484～+2)克隆到含荧光素酶报告基因的质粒载体pGL3-Enhancer中，得到高表达的质粒484-luc。然后把484-luc转染到B淋巴细胞IM-9中，在无血清培养56hr后，分别将不同浓度的激素或细胞因子加入培养液中，4hr后收集细胞，测定荧光素酶活性。发现GHRH(0.001nM)和IL-1β(10～1000U／ml)对B淋巴细胞中GH基因转录起抑制作用，而IGF-1(1～100nM)、IL-2(10～1000U／ml)、IL-4(10ng／ml)、TNF-α(100～1000U／ml)和IFN-γ(10～1000U／ml)起促进作用。其中IFN-γ作用最明显，它能够剂量依赖性地促进B淋巴细胞中GH基因的转录，其浓度为1000U／ml时，GH基因转录水平增加51％。SMS、IL-5、IL-6和GH对B淋巴细胞中GH基因的转录没有显著影响。这说明B淋巴细胞中GH基因转录受激素和细胞因子的调节，但其调节机制与垂体GH基因不同。 2．B淋巴细胞中GH基因的基础转录与细胞内信息传递途径的关系：将484-luc转染到B淋巴细胞中，无血清培养56hr后，用不同浓度的细胞内信息传递途径的抑制剂或激动剂处理细胞，发现MAPK抑制剂PD98059(40μM)能够显著抑制GH基因的转录(抑制48％)。这说明MAPK途径对B淋巴细胞中GH更多还原

【Abstract】 Growth hormone (GH) has important stimulating effect on immune function and its association with immune function aroused great investigating interests. GH and its receptor could be expressed by human and rodent lymphocytes. Lymphocytic GH could affect the function of lymphocyte in an autocrine and/or paracrine way. Now the control mechanism of GH gene expression in lymphocyte is unclear. It was found that some hormones could affect lymphocyte GH secretion. Cytokines have significant impact on lymphocytic growth, differentiation and functions. There are few reports about the effects of hormones and cytokines on lymphocyte GH gene transcription. We addressed this question with the method of luciferase reporter gene. In addition, we explored the association of GH gene transcription and intracellular signaling transduction pathway, the relative transcription activity of various deletions of GH gene promoter, and the association of GH gene transcription with pituitary specific transcription factor Pit-1, in B-lymphocytes.1. The effects of cytokines and hormones on GH gene transcription in B-lymphocyte. We insert GH gene promoter (-484~+2 bp) into luciferase reporter gene vector pGL3-Enhancer. The recombinant plasmid was abbreviated as 484-luc. Then 484-luc was transfected into B-lymphocyte. Fifty-six hours after transfection, B-lymphocytes were treated with various hormones and cytokines at different concentration. Four hours later, cells were harvested and lysed to measure luciferase activity. The results demonstrated that GHRH (0.001 nM) and IL-1β (10~1000U/ml) significantly inhibited, whereas IGF-1 (1-100 nM), IL-2 (10-1000 U/ml), IL-4 (10 ng/ml), TNF-α (100-1000 U/ml) and IFN-γ (10-1000 U/ml) significantly stimulated GH transcription in B-lymphocytes. Among all the observed hormones and cytokines, IFN-γ had most prominent effect. It increased GH gene transcription in B-lymphocytes in a dose-dependent manner. When it’s concentration was 1000 U/ml, GH gene transcription increased by 51 % as compared with control. SMS (a somatostatin analogue), IL-5, and IL-6 had no significant effect on GH gene transcription in B-lymphocyte. Above results suggested that the更多还原