淋巴细胞来源的儿茶酚胺对T辅助细胞分化的影响

发布时间：2018-09-14 10:03

【摘要】：目的：随着神经—内分泌—免疫相互作用研究的不断深入,人们发现大鼠和人的免疫细胞均能表达酪氨酸羟化酶(tyrosine hydroxylase, TH)即儿茶酚胺(catecholamine, CAs)合成的限速酶,随后的研究也证实了淋巴细胞自身能分泌儿茶酚胺,并对淋巴细胞的增殖和凋亡产生影响,并在此基础上初步探讨了其作用机制。T淋巴细胞是高度异质性的群体,CD4+T细胞是T细胞中最多的一类亚群,本课题以CD4+T细胞为研究对象,探讨CD4+T细胞自身分泌的儿茶酚胺对于Th细胞分化的影响,从而为神经—内分泌—免疫调节网络增加新的内容。方法：取小鼠肠系膜淋巴结,常规培养淋巴细胞,用磁珠分选法分离和纯化CD4+T淋巴细胞。用逆转录—聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)法检测CD4+T淋巴细胞TH的表达。用Western blot方法检测CD4+T淋巴细胞TH的表达。构建四个针对TH(NM_009377.1)基因的pcDNATM6.2-GW/miRNA干扰质粒,用AMAXA neucleofector技术将干扰质粒转染CD4+T淋巴细胞,应用RNA干扰技术抑制小鼠CD4+T细胞TH的表达,用(?) real-time PCR方法和western-blot方法分别检测TH基因和蛋白水平的沉默效果,高效液相色谱法-电化学检测法检测TH基因沉默后胞内CAs含量变化,用流式细胞术检测TH基因沉默后的CD4+T细胞细胞内IL-4和IFN-y的含量。结果：淋巴细胞经分离和纯化后,CD4+T细胞纯度达到98.2%, RT-PCR方法和Western blot方法证明小鼠CD4+T淋巴细胞表达THmRNA以及TH蛋白。构建的4对针对TH基因miRNA表达载体pcDNATM6.2-GW/miRNA经测序鉴定,证明构建成功。AMAXA neucleofector技术转染pcDNATM6.2-GW/miRNA进入CD4+T淋巴细胞,转染效率达60%。实时荧光定量PCR检测小鼠CD4+T细胞转染干扰质粒后TH的干扰抑制效率,pcDNATM6.2-GW/miRNA3, pcDNATM6.2-GW/miRNA4的干扰作用可达70%左右,其中以miRNA4插入片段的抑制作用最为明显。Western blot方法检测干扰质粒的干扰抑制效果,也达70%左右,与real-time PCR结果相一致,以pcDNATM6.2-GW/miRNA4插入片段抑制作用最为明显。高效液相色谱-电化学检测法检测结果证明TH基因沉默后CD4+T淋巴细胞胞内合成儿茶酚胺含量明显降低,流式细胞术检测细胞内细胞因子结果显示TH基因沉默,导致了CD4+T淋巴细胞胞内IL-4含量的下降,而对胞内IFN-γ的含量无明显影响,Th1/Th2比例上升。结论：小鼠CD4+T细胞能表达TH, TH基因的沉默抑制了淋巴细胞来源的CAs的合成,Th细胞来源的CAs促进Th细胞向着Th2方向分化。
[Abstract]:Aim: with the development of neuroendocrine immune interaction, it has been found that both rat and human immune cells can express tyrosine hydroxylase (tyrosine hydroxylase, TH), which is the rate-limiting enzyme of catecholamine (catecholamine, CAs) synthesis. Subsequent studies have also confirmed that lymphocytes secrete catecholamine and have an effect on lymphocyte proliferation and apoptosis. On the basis of this, we preliminarily discussed its mechanism. The T lymphocyte is a highly heterogeneous population. CD4 T cells are the most subsets of T cells. In this study, CD4 T cells were taken as the research object. To investigate the effect of catecholamines secreted by CD4 T cells on the differentiation of Th cells, so as to add new contents to the neuroendocrine immune regulatory network. Methods: CD4 T lymphocytes were isolated and purified from mouse mesenteric lymph nodes by routine culture. Reverse transcriptase polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) was used to detect the expression of TH in CD4 T lymphocytes. The expression of TH in CD4 T lymphocytes was detected by Western blot method. Four pcDNATM6.2-GW/miRNA interference plasmids targeting TH (NM_009377.1) gene were constructed. The interfering plasmids were transfected into CD4 T lymphocytes by AMAXA neucleofector technique. RNA interference technique was used to inhibit the TH expression of CD4 T cells in mice. The silencing effect of TH gene and protein level was detected by (?) real-time PCR method and western-blot method, and the change of intracellular CAs content after TH gene silencing was detected by high performance liquid chromatography (HPLC) -electrochemical detection method. The content of IL-4 and IFN-y in CD4 T cells after TH gene silencing was detected by flow cytometry. Results: the purity of CD 4 T cells reached 98.2% after isolation and purification. The expression of THmRNA and TH protein in mouse CD4 T lymphocytes was confirmed by RT-PCR and Western blot methods. Four pairs of miRNA expression vector pcDNATM6.2-GW/miRNA targeting TH gene were identified by sequencing. It was proved that pcDNATM6.2-GW/miRNA was successfully transfected into CD4 T lymphocytes by AMAXA neucleofector technique, and the transfection efficiency was 60%. Real-time fluorescence quantitative PCR was used to detect the interference inhibition efficiency of TH after transfection of mouse CD4 T cells and the interference efficiency of pcDNATM6.2-GW / miRNA3.The interference effect of pcDNATM6.2-GW/miRNA4 could reach about 70%. Among them, the inhibitory effect of miRNA4 insert fragment was the most obvious. Western blot method was used to detect the interference inhibition effect of interference plasmid, which was about 70%, which was consistent with the result of real-time PCR. The inhibition effect of pcDNATM6.2-GW/miRNA4 insert fragment was the most obvious. The results of high performance liquid chromatography-electrochemical detection showed that the intracellular synthesis of catecholamine in CD4 T lymphocytes was significantly decreased after TH gene silencing, and the intracellular cytokines were detected by flow cytometry. TH gene silencing was observed by flow cytometry. It resulted in the decrease of IL-4 content in CD4 T lymphocytes, but the increase of Th1 / Th2 ratio was not significantly affected by the content of IFN- 纬 in T lymphocytes. Conclusion: the silencing of the expression of TH, TH gene in mouse CD4 T cells inhibits the synthesis of CAs derived from lymphocytes and CAs derived from Th cells promotes the differentiation of Th cells towards Th2.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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