淋巴細(xì)胞來(lái)源的兒茶酚胺對(duì)T輔助細(xì)胞分化的影響
發(fā)布時(shí)間：2018-09-14 10:03
【摘要】：目的：隨著神經(jīng)—內(nèi)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(xì)胞均能表達(dá)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(shí)了淋巴細(xì)胞自身能分泌兒茶酚胺,并對(duì)淋巴細(xì)胞的增殖和凋亡產(chǎn)生影響,并在此基礎(chǔ)上初步探討了其作用機(jī)制。T淋巴細(xì)胞是高度異質(zhì)性的群體,CD4+T細(xì)胞是T細(xì)胞中最多的一類亞群,本課題以CD4+T細(xì)胞為研究對(duì)象,探討CD4+T細(xì)胞自身分泌的兒茶酚胺對(duì)于Th細(xì)胞分化的影響,從而為神經(jīng)—內(nèi)分泌—免疫調(diào)節(jié)網(wǎng)絡(luò)增加新的內(nèi)容。 方法：取小鼠腸系膜淋巴結(jié),常規(guī)培養(yǎng)淋巴細(xì)胞,用磁珠分選法分離和純化CD4+T淋巴細(xì)胞。用逆轉(zhuǎn)錄—聚合酶鏈反應(yīng)(reverse transcription-polymerase chain reaction, RT-PCR)法檢測(cè)CD4+T淋巴細(xì)胞TH的表達(dá)。用Western blot方法檢測(cè)CD4+T淋巴細(xì)胞TH的表達(dá)。構(gòu)建四個(gè)針對(duì)TH(NM_009377.1)基因的pcDNATM6.2-GW/miRNA干擾質(zhì)粒,用AMAXA neucleofector技術(shù)將干擾質(zhì)粒轉(zhuǎn)染CD4+T淋巴細(xì)胞,應(yīng)用RNA干擾技術(shù)抑制小鼠CD4+T細(xì)胞TH的表達(dá),用(?) real-time PCR方法和western-blot方法分別檢測(cè)TH基因和蛋白水平的沉默效果,高效液相色譜法-電化學(xué)檢測(cè)法檢測(cè)TH基因沉默后胞內(nèi)CAs含量變化,用流式細(xì)胞術(shù)檢測(cè)TH基因沉默后的CD4+T細(xì)胞細(xì)胞內(nèi)IL-4和IFN-y的含量。 結(jié)果：淋巴細(xì)胞經(jīng)分離和純化后,CD4+T細(xì)胞純度達(dá)到98.2%, RT-PCR方法和Western blot方法證明小鼠CD4+T淋巴細(xì)胞表達(dá)THmRNA以及TH蛋白。構(gòu)建的4對(duì)針對(duì)TH基因miRNA表達(dá)載體pcDNATM6.2-GW/miRNA經(jīng)測(cè)序鑒定,證明構(gòu)建成功。AMAXA neucleofector技術(shù)轉(zhuǎn)染pcDNATM6.2-GW/miRNA進(jìn)入CD4+T淋巴細(xì)胞,轉(zhuǎn)染效率達(dá)60%。實(shí)時(shí)熒光定量PCR檢測(cè)小鼠CD4+T細(xì)胞轉(zhuǎn)染干擾質(zhì)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(dá)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(cè)干擾質(zhì)粒的干擾抑制效果,也達(dá)70%左右,與real-time PCR結(jié)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(xué)檢測(cè)法檢測(cè)結(jié)果證明TH基因沉默后CD4+T淋巴細(xì)胞胞內(nèi)合成兒茶酚胺含量明顯降低,流式細(xì)胞術(shù)檢測(cè)細(xì)胞內(nèi)細(xì)胞因子結(jié)果顯示TH基因沉默,導(dǎo)致了CD4+T淋巴細(xì)胞胞內(nèi)IL-4含量的下降,而對(duì)胞內(nèi)IFN-γ的含量無(wú)明顯影響,Th1/Th2比例上升。結(jié)論：小鼠CD4+T細(xì)胞能表達(dá)TH, TH基因的沉默抑制了淋巴細(xì)胞來(lái)源的CAs的合成,Th細(xì)胞來(lái)源的CAs促進(jìn)Th細(xì)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(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：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(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
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