【作者】 黄彦；

【导师】 邱一华；

【作者基本信息】 南通大学 ， 生理学， 2008， 硕士

【摘要】 目的:随着神经-内分泌-免疫相互作用研究的不断深入,目前已知在淋巴细胞上具有α-和β-肾上腺素受体,并且它们都参与调节免疫细胞功能。然而,尽管已有文献报道淋巴细胞上也存在多巴胺(dopamine, DA)受体,但关于T淋巴细胞上存在哪种亚型DA受体,以及这些受体在介导T细胞功能调节中的作用尚不十分清楚。因此,本研究旨在一方面提供T淋巴细胞表达DA两类亚型受体――D1-like受体(包括D1和D5受体)和D2-like受体(包括D2、D3、D4受体)的证据,另一方面探讨D1-like受体和D2-like受体在介导T细胞功能调节中的作用及其信号转导分子,从而为神经-内分泌-免疫调节网络增加新内容。方法:取小鼠肠系膜淋巴结细胞,用尼龙毛柱粘附法分离和纯化T淋巴细胞,应用流式细胞术检测T细胞的纯度。用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)法检测T淋巴细胞的各种亚型DA受体mRNA表达。淋巴细胞用各种浓度的D1-like受体激动剂、拮抗剂或D2-like受体激动剂、拮抗剂孵育48 h,然后应用噻唑兰(methyl-thiazole-tetrazolium, MTT)比色法检测淋巴细胞对有丝分裂原――刀豆蛋白A(concanavalin A, Con A)诱导的增殖反应;应用流式蛋白分析系统测定经Con A刺激的淋巴细胞培养物的上清液中细胞因子干扰素-γ(interferon-γ,IFN-γ)、肿瘤坏死因子(tumornecrosisfactor,TNF)和白介素-4(interleukin-4,IL-4)的浓度;应用125I-环一磷酸腺苷(125I-cAMP)放射免疫法检测淋巴细胞内环一磷酸腺苷(cyclic adenosine monophosphate,cAMP)的含量。结果:肠系膜淋巴结细胞经分离和纯化后,获得的CD3+淋巴细胞(即T淋巴细胞)在肠系膜淋巴结细胞中的百分比为93.8±0.53%,明显高于未经分离和纯化的肠系膜淋巴结细胞中的T细胞百分比74.1±0.95%,说明用上述方法已获得纯化的T淋巴细胞。T淋巴细胞可表达所有DA亚型受体mRNA,包括D1-like受体的D1和D5受体,及D2-like受体的D2、D3、D4受体。淋巴细胞分别用5个浓度的D1-like受体激动剂SKF38393(10-9～10-5 M)处理后,细胞对Con A诱导的增殖反应均没有发生明显的改变。用Con A刺激的淋巴细胞经SKF38393(10-8 M)处理后,IFN-γ的产生显著低于未经SKF38393处理的淋巴细胞,但TNF和IL-4的产生没有明显改变。D1-like受体拮抗剂SCH23390(10-7 M)能阻断激动剂SKF38393(10-8 M)对淋巴细胞产生IFN-γ的抑制作用。D1-like受体激动剂、拮抗剂或激动剂与拮抗剂一起处理淋巴细胞,均没有明显影响淋巴细胞内cAMP的含量。上述结果说明,D1-like受体在介导T细胞功能调节中不起主导作用。然而,淋巴细胞分别用5个浓度的D2-like受体激动剂quinpirole(10-9～10-5 M)处理后,细胞对Con A诱导的增殖反应均明显减弱。用不同浓度的D2-like受体拮抗剂haloperidol(10-10～10-7 M)与激动剂quinpirole(10-8 M或10-7 M)一起处理淋巴细胞,haloperidol呈现浓度依赖性地阻断quinpirole对T细胞增殖的抑制作用。quinpirole(10-8 M)可抑制Con A刺激的T细胞生成IFN-γ和TNF,但促进T细胞生成IL-4。haloperidol(10-7 M)能阻断quinpirole抑制T细胞生成IFN-γ和TNF的作用,及阻断quinpirole促进T细胞生成IL-4的作用。上述结果充分说明D2-like受体介导调节T细胞的功能。此外,quinpirole(10-8 M和10-7 M)可导致Con A激活的T细胞内cAMP含量明显减少,haloperidol(10-8 M和10-7 M)则可阻断quinpirole减少淋巴细胞内cAMP的作用,提示cAMP参与D2-like受体介导的T淋巴细胞功能调节。结论:T淋巴细胞能表达各种DA亚型受体――D1、D5受体(即D1-like受体)及D2、D3、D4受体(即D2-like受体)。在D1-like和D2-like两型DA受体中,D2-like受体是介导T淋巴细胞功能调节的主要受体,它的激活能抑制T淋巴细胞的增殖、减少细胞因子IFN-γ和TNF的生成及增加IL-4的生成,D2-like受体的这些作用经淋巴细胞内第二信使cAMP介导。更多还原

【Abstract】 Objectives: With the increasing studies on neural-endocrine-immune interaction, lymphocytes have been shown to haveα- andβ-adrenoreceptors and the receptors participate in mediating the regulation of immune cell function. However, although some reports have demonstrated that dopamine (DA) receptors exist in lymphocytes, which DA receptor subtypes are on the T lymphocytes and what roles of the DA receptor subtypes in mediating the modulation of T cell function are less known. Thus, in the present study, on the one hand we provided the further evidence for the expressions of two subtypes of DA receptors, D1-like receptors (including D1 and D5 receptors) and D2-like receptors (including D2, D3 and D4 receptors), in T lymphocytes, and on the other hand we explored the roles of D1-like and D2-like receptors in mediating the modulation of T cell function and signal transduction molecules involved in the modulation in order to extend the comprehension of neural-endocrine-immune regulatory network. Methods: Lymphocytes were separated from the mesenteric lymph nodes of mice. T cells were purified by using a nylon wool column. The purity of the T lymphocytes purified was determined by flow cytometric assay. Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression of each subtype of DA receptor mRNA in the purified T cells. The lymphocytes from the mesenteric lymph nodes of mice were incubated with concanavalin A (Con A) and treated with selective D1-like receptor agonist (SKF38393) and antagonist (SCH23390), or with selective D2-like receptor agonist (quinpirole) and antagonist (haloperidol) for 48 h. Colorimetric assay of methylthiazol tetrazolium bromide (MTT) was employed to measure the proliferative response of the lymphocytes to Con A. Cytometric bead array was used to examine the levels of cytokines interferon-γ(IFN-γ), tumor necrosis factor (TNF) and interleukin-4 (IL-4) in the supernatants of the Con A-stimulated lymphocyte cultures. The content of cAMP in the Con A-activated lymphocytes was measured by 125I-cAMP radioimmunoassay. Results: The percentage of purified T lymphocytes (CD3+) in the mesenteric lymph node cells was 93.8±0.53%, which was significantly higher than 74.1±0.95% of T cells that were not purified. The purified T lymphocytes expressed the all subtypes of DA receptor mRNA, including D1 and D5 of D1-like receptors, as well as D2, D3 and D4 of D2-like receptors. After the lymphocytes were treated with D1-like receptor agonist SKF38393 (10-9 to 10-5 M), the proliferative response of the lymphocytes to Con A did not exhibit an evident change. Similarly, the production of TNF and IL-4 by the con A-stimulated lymphocytes that were treated with SKF38393(10-8 M)did not show remarkable change, but the IFN-γproduction by the lymphocytes treated with SKF38393 was notably lower than that of SKF38393-untreated lymphocytes. D1-like receptor antagonist SCH23390 (10-7 M) blocked the inhibitory effect of agonist SKF38393 on the IFN-γproduction by the Con A-stimulated lymphocytes. The content of cAMP in the Con A-activated lymphocytes did not exhibit a notable change no matter what treatments were used with D1-like receptor agonist and antagonist. However, the treatment of lymphocytes with D2-like receptor agonist quinpirole(10-9 to 10-5 M)led to a dramatic attenuation in the proliferative response of the lymphocytes to Con A. D2-like receptor antagonist haloperidol ( 10-10 to 10-7 M ) represented a concentration-dependent blockage of the suppressive effect of quinpirole on the Con A-induced lymphocyte proliferation. Furthermore, quinpirole (10-8 M) reduced the production of IFN-γand TNF, but increased the IL-4 production by the Con A-stimulated lymphocytes. Haloperidol (10-7 M) blocked the quinpirole-induced decrease in IFN-γand TNF levels and the increase in IL-4 production. The cAMP content in the lymphocytes was found decreased after the lymphocytes were treated with quinpirole (10-8 and 10-7 M), and haloperidol (10-8 and 10-7 M) blocked the decrease in cAMP content induced by quinpirole. Conclusions: T lymphocytes can express all the subtypes of DA receptors, including D1 and D5 receptors (i.e., D1-like receptors) and D2, D3, and D4 receptors (i.e., D2-like receptors). Of DA D1-like and D2-like receptors, D2-like receptors are more important in mediating the modulation of T cell function. The activation of D2-like receptors results in an inhibition of T cell proliferation, a decrease in IFN-γand TNF production and an increase in IL-4 level. The immunomodulation mediated by D2-like receptors may be performed by the second messenger cAMP in lymphocytes.更多还原