淋巴細胞來源的兒茶酚胺對T輔助細胞分化的影響
發(fā)布時間：2018-09-14 10:03
【摘要】：目的：隨著神經(jīng)—內分泌—免疫相互作用研究的不斷深入,人們發(fā)現(xiàn)大鼠和人的免疫細胞均能表達酪氨酸羥化酶(tyrosine hydroxylase, TH)即兒茶酚胺(catecholamine, CAs)合成的限速酶,隨后的研究也證實了淋巴細胞自身能分泌兒茶酚胺,并對淋巴細胞的增殖和凋亡產生影響,并在此基礎上初步探討了其作用機制。T淋巴細胞是高度異質性的群體,CD4+T細胞是T細胞中最多的一類亞群,本課題以CD4+T細胞為研究對象,探討CD4+T細胞自身分泌的兒茶酚胺對于Th細胞分化的影響,從而為神經(jīng)—內分泌—免疫調節(jié)網(wǎng)絡增加新的內容。 方法：取小鼠腸系膜淋巴結,常規(guī)培養(yǎng)淋巴細胞,用磁珠分選法分離和純化CD4+T淋巴細胞。用逆轉錄—聚合酶鏈反應(reverse transcription-polymerase chain reaction, RT-PCR)法檢測CD4+T淋巴細胞TH的表達。用Western blot方法檢測CD4+T淋巴細胞TH的表達。構建四個針對TH(NM_009377.1)基因的pcDNATM6.2-GW/miRNA干擾質粒,用AMAXA neucleofector技術將干擾質粒轉染CD4+T淋巴細胞,應用RNA干擾技術抑制小鼠CD4+T細胞TH的表達,用(?) real-time PCR方法和western-blot方法分別檢測TH基因和蛋白水平的沉默效果,高效液相色譜法-電化學檢測法檢測TH基因沉默后胞內CAs含量變化,用流式細胞術檢測TH基因沉默后的CD4+T細胞細胞內IL-4和IFN-y的含量。 結果：淋巴細胞經(jīng)分離和純化后,CD4+T細胞純度達到98.2%, RT-PCR方法和Western blot方法證明小鼠CD4+T淋巴細胞表達THmRNA以及TH蛋白。構建的4對針對TH基因miRNA表達載體pcDNATM6.2-GW/miRNA經(jīng)測序鑒定,證明構建成功。AMAXA neucleofector技術轉染pcDNATM6.2-GW/miRNA進入CD4+T淋巴細胞,轉染效率達60%。實時熒光定量PCR檢測小鼠CD4+T細胞轉染干擾質粒后TH的干擾抑制效率,pcDNATM6.2-GW/miRNA3, pcDNATM6.2-GW/miRNA4的干擾作用可達70%左右,其中以miRNA4插入片段的抑制作用最為明顯。Western blot方法檢測干擾質粒的干擾抑制效果,也達70%左右,與real-time PCR結果相一致,以pcDNATM6.2-GW/miRNA4插入片段抑制作用最為明顯。高效液相色譜-電化學檢測法檢測結果證明TH基因沉默后CD4+T淋巴細胞胞內合成兒茶酚胺含量明顯降低,流式細胞術檢測細胞內細胞因子結果顯示TH基因沉默,導致了CD4+T淋巴細胞胞內IL-4含量的下降,而對胞內IFN-γ的含量無明顯影響,Th1/Th2比例上升。結論：小鼠CD4+T細胞能表達TH, TH基因的沉默抑制了淋巴細胞來源的CAs的合成,Th細胞來源的CAs促進Th細胞向著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- 緯 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.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
本文編號：2242392
[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- 緯 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.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
【參考文獻】
相關期刊論文 前10條
1 劉加洪,王英年,趙云峰,姚艷紅,張克佳;Th1-Th2平衡失調與人類疾病關系及其相關治療研究現(xiàn)狀[J];青島大學醫(yī)學院學報;2002年04期
2 金秀東;關艷中;張書捷;徐滿英;岳文杰;;酚妥拉明阻斷去甲腎上腺素對嗎啡成癮大鼠束旁核痛反應神經(jīng)元電活動的影響[J];南方醫(yī)科大學學報;2008年02期
3 王紅艷;自身免疫性疾病中T_H1/T_H2細胞平衡[J];國外醫(yī)學(免疫學分冊);2000年06期
4 陳女燕,蔣雨平,朱萍;帕金森病外周血T細胞亞群的研究[J];中國臨床神經(jīng)科學;2004年04期
5 邱一華,彭聿平,王建軍;兒茶酚胺調節(jié)免疫功能的細胞和分子機制[J];生理科學進展;2003年04期
6 朱紅濤;周敬群;;兒茶酚胺對T細胞和B細胞的調節(jié)作用[J];臨床和實驗醫(yī)學雜志;2007年01期
7 ;Effect of catecholamines on IL-2 production and NK cytotoxicity of rats in vitro[J];Acta Pharmacologica Sinica;2004年10期
8 姜建蘭;彭聿平;邱一華;王建軍;;淋巴細胞合成的內源性兒茶酚胺對T細胞增殖功能的影響[J];中國應用生理學雜志;2009年01期
9 彭聿平，，邱一華，張清泉，王健鶴;去甲腎上腺素對T淋巴細胞增殖的影響[J];中國應用生理學雜志;1995年01期
10 許繼平,李大年;帕金森病的藥物治療研究進展[J];中華神經(jīng)科雜志;1997年03期
本文編號：2242392
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2242392.html
最近更新
教材專著
