Liu Yan, Huang Yan, Wang Xiao-Qin, Peng Yu-Ping, Qiu Yi-Hua
Department of Physiology, School of Medicine, Nantong University, Nantong, China.
Neuro Endocrinol Lett. 2012;33(6):643-50.
We explored effect of gene silencing of tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of catecholamines (CAs), in CD4+ T cells on differentiation and function of helper T (Th) cells to provide more evidence for functional significance of lymphocyte-derived CAs.
CD4+ T lymphocytes were isolated and purified from the mesenteric lymph nodes of mice. Recombinant TH miRNA expression vector (pcDNA6.2-GW/EmGFPmiR-TH) was constructed and transfected into concanavalin A (Con A)-activated CD4+ T lymphocytes using nucleofection technology. After incubated for 48 h, these cells were detected for TH gene and protein expression and CA content. Simultaneously, percentage of interferon-γ (IFN-γ)- and interleukin-4 (IL-4)-producing cells and levels of IL-2, IFN-γ, tumor necrosis factor (TNF), IL-4 and IL-5 in culture supernatants of Con A-stimulated CD4+ T cells were examined by flow cytometric analysis.
CD4+ T lymphocytes with TH RNAi expressed less TH mRNA and protein and synthesized less CAs including norepinephrine, epinephrine and dopamine than control cells with mock transfection. The silencing of TH gene in CD4+ T lymphocytes reduced percentage of IL-4-producing cells and elevated ratio of IFN-γ-producing cells to IL-4-producing cells, although it did not alter proportion of IFN-γ-producing cells. The Th1 cytokines, IL-2, IFN-γ and TNF, were increased, but the Th2 cytokines, IL-4 and IL-5, were decreased in the culture supernatants of Con A-stimulated CD4+ T lymphocytes that were transfected with TH miRNA.
TH gene silencing attenuates TH expression and CA synthesis in CD4+ T lymphocytes and promotes polarization of differentiation and function towards Th1 cells.
我们探讨了在CD4+ T细胞中，酪氨酸羟化酶（TH，儿茶酚胺合成的限速酶）基因沉默对辅助性T（Th）细胞分化和功能的影响，为淋巴细胞源性儿茶酚胺的功能意义提供更多证据。
从小鼠肠系膜淋巴结中分离并纯化CD4+ T淋巴细胞。构建重组TH miRNA表达载体（pcDNA6.2-GW/EmGFPmiR-TH），并使用核转染技术将其转染到刀豆蛋白A（Con A）激活的CD4+ T淋巴细胞中。孵育48小时后，检测这些细胞的TH基因和蛋白表达以及儿茶酚胺含量。同时，通过流式细胞术分析检测Con A刺激的CD4+ T细胞培养上清液中产生干扰素-γ（IFN-γ）和白细胞介素-4（IL-4）的细胞百分比以及IL-2、IFN-γ、肿瘤坏死因子（TNF）、IL-4和IL-5的水平。
与mock转染的对照细胞相比，具有TH RNAi的CD4+ T淋巴细胞表达的TH mRNA和蛋白较少，合成的包括去甲肾上腺素、肾上腺素和多巴胺在内的儿茶酚胺也较少。CD4+ T淋巴细胞中TH基因的沉默降低了产生IL-4的细胞百分比，并提高了产生IFN-γ的细胞与产生IL-4的细胞的比例，尽管它没有改变产生IFN-γ的细胞比例。在用TH miRNA转染的Con A刺激的CD4+ T淋巴细胞培养上清液中，Th1细胞因子IL-2、IFN-γ和TNF增加，而Th2细胞因子IL-4和IL-5减少。
TH基因沉默减弱了CD4+ T淋巴细胞中TH的表达和儿茶酚胺的合成，并促进分化和功能向Th1细胞极化。
