本文關(guān)鍵詞： γδT細(xì)胞 Vδ亞群 結(jié)核病人 RTPCR 流式細(xì)胞儀　出處：《蚌埠醫(yī)學(xué)院》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景：結(jié)核病是嚴(yán)重危害人類(lèi)健康的常見(jiàn)感染性疾病之一，但關(guān)于機(jī)體抗結(jié)核感染的免疫機(jī)制尚未完全闡明。以往研究認(rèn)為，αβ T細(xì)胞中CD4+和CD8+T細(xì)胞，以及巨噬細(xì)胞在抗結(jié)核免疫中發(fā)揮關(guān)鍵性作用。近年來(lái)很多研究結(jié)果證實(shí)γδ T細(xì)胞在抗結(jié)核桿菌感染免疫中也發(fā)揮重要作用。γδ T細(xì)胞是與αβ T細(xì)胞有不同特征的獨(dú)特T細(xì)胞，許多研究表明γδ T細(xì)胞在抗感染免疫、抗腫瘤免疫中發(fā)揮重要作用，同時(shí)γδ T細(xì)胞在調(diào)節(jié)特異性免疫應(yīng)答、自身免疫性疾病和變態(tài)反應(yīng)性疾病的發(fā)生均有重要的參與作用。γδ T細(xì)胞可產(chǎn)生多種細(xì)胞因子和趨化因子，在抗感染免疫，特別是在抗結(jié)核桿菌感染的早期免疫中可能發(fā)揮重要作用。γδ T細(xì)胞根據(jù)δ鏈V區(qū)不同可分為8個(gè)亞群（Vδ1至Vδ8），正常人以為Vδ2亞群為主。有研究顯示，在某些疾病患者γδ T細(xì)胞中Vδ1和Vδ2亞群的比例明顯改變。本研究室的前期研究中也觀察到同樣現(xiàn)象。我們關(guān)注的科學(xué)問(wèn)題是：健康成年人與結(jié)核病人γδ T細(xì)胞的Vδ1-8各亞群分布情況究竟是怎樣的，健康成年人與結(jié)核病人是否有明顯差異？尤其是Vδ1亞群和Vδ2亞群。 目的：調(diào)查健康成人外周血中Vδ1-8各亞群的分布情況和結(jié)核病人外周血中Vδ1-8各亞群的分布改變，通過(guò)研究對(duì)比，探討γδ T細(xì)胞Vδ1-8各亞群，特別是Vδ1亞群和Vδ2亞群在結(jié)核感染中可能發(fā)揮的不同作用。 方法：抽取一定數(shù)量健康成人和結(jié)核病人空腹靜脈血于肝素鈉抗凝管中備用，取試管與不完全1640培養(yǎng)液混勻，用人淋巴細(xì)胞分離液經(jīng)密度梯度離心法獲取外周血單個(gè)核細(xì)胞。用磷酸鹽緩沖液洗滌后，調(diào)整細(xì)胞濃度5×106/ml－1×107/ml。 流式細(xì)胞儀檢測(cè)法取上述細(xì)胞懸液，分別加入抗人CD3-FITC，抗人CD3-APC，抗人Vδ1-PE，抗人γδ-TC，抗人Vδ1-FITC，抗人Vδ2-PE表面染色后，用流式細(xì)胞儀檢測(cè)。數(shù)據(jù)分析采用Cell Quest軟件，計(jì)算Vδ1+，Vδ2+細(xì)胞以及Vδ1－/Vδ2－(即Vδ3～8+)細(xì)胞各自在γδ T細(xì)胞中的百分比。 熒光定量RT－PCR檢測(cè)法取上述細(xì)胞懸液用Trizol裂解后提取RNA并用超微量核酸蛋白檢測(cè)儀鑒定RNA質(zhì)量，，合格者用RevertAid First Strand cDNASynthesis Kit試劑盒按標(biāo)準(zhǔn)加樣后在PCR儀上逆轉(zhuǎn)錄得到cDNA，再將cDNA按標(biāo)準(zhǔn)加入SYBR Green Reagents熒光試劑，放入實(shí)時(shí)熒光定量PCR儀反應(yīng)，反應(yīng)參照基因?yàn)楦视腿?3-磷酸脫氫酶。所得數(shù)據(jù)將δ1-8基因循環(huán)數(shù)與內(nèi)參的差值經(jīng)過(guò)2-換算后得到各基因相對(duì)表達(dá)量，并計(jì)算均數(shù)和標(biāo)準(zhǔn)差。 結(jié)果：1.36例健康成年人中，流式細(xì)胞儀檢測(cè)，大多數(shù)人γδ T細(xì)胞δ2亞群所占比例最高；實(shí)時(shí)熒光定量RT-PCR法檢測(cè)Vδ1-8基因，δ2基因相對(duì)表達(dá)量為0.0955±0.006，占總γδ T細(xì)胞表達(dá)量的57.96%。其他基因按相對(duì)表達(dá)量由多到少排列，依次為δ3、δ1、δ7、δ8、δ4、δ5、δ6。 2.10例結(jié)核病人當(dāng)中，流式細(xì)胞儀檢測(cè)，大多數(shù)人γδ T細(xì)胞主要以δ1亞群為主；實(shí)時(shí)熒光定量RT-PCR法檢測(cè)δ1-8基因，δ1基因相對(duì)表達(dá)量為0.01723±0.0001，占總γδ T細(xì)胞表達(dá)量的46.60%。δ2基因相對(duì)表達(dá)量為0.0042±0.0007，僅占總γδ T細(xì)胞表達(dá)量的11.44%。其他基因按相對(duì)表達(dá)量由多到少排列，依次為δ5、δ6、δ4、δ7、δ3、δ8。 3.統(tǒng)計(jì)學(xué)驗(yàn)證相關(guān)系數(shù)，健康成年人組的Vδ1組，Vδ2組，Vδ1－/Vδ2－(即Vδ3-8+)組R2在0.941到0.985間，結(jié)核病人組的Vδ1組，Vδ2組，Vδ3-8組R2在0.967到0.990間，(P0.05)。 結(jié)論：1.流式細(xì)胞術(shù)和實(shí)時(shí)定量PCR技術(shù)檢測(cè)發(fā)現(xiàn)，36例健康成年人γδ T細(xì)胞亞群中，以Vδ2亞群比例最高（占總γδ T細(xì)胞57.96%），其他亞群的比例由多到少依次為δ3、δ1、δ7、δ8、δ4、δ5、δ6。 2.與健康成年人不同，10例結(jié)核病人的γδ T細(xì)胞主要以δ1亞群為主（占總γδT細(xì)胞46.60%），其他亞群的比例由多到少依次為δ5、δ6、δ2、δ4、δ7、δ3、δ8。 3.流式細(xì)胞儀檢測(cè)法與實(shí)時(shí)定量PCR檢測(cè)法呈較強(qiáng)的線(xiàn)性正相關(guān)關(guān)系，統(tǒng)計(jì)學(xué)驗(yàn)證健康成年人組和結(jié)核病人組不同亞群，相關(guān)系數(shù)R2在0.941到0.990間。 4.結(jié)核病人Vδ2細(xì)胞亞群較健康成年人明顯減少，而Vδ1細(xì)胞亞群相對(duì)增高。推斷該Vδ2細(xì)胞亞群在結(jié)核感染中被消耗所導(dǎo)致。
[Abstract]:Background: tuberculosis is one of the most common infectious diseases serious harm to human health, but on the immune mechanism of anti tuberculosis infection has not been fully elucidated. Previous studies suggest that CD4+ and CD8+T cells of alpha beta T cells, and macrophages in anti tuberculosis immunity play a key role. In recent years, many research results show that gamma delta T cells in anti tuberculosis infection also plays an important role in immune. T cells have different characteristics and unique alpha beta T cells T cells, many studies have shown that gamma delta T cells in anti infection immunity, antitumor immunity and play an important role at the same time, gamma delta T cells in regulating specific immune response. An important role has occurred in autoimmune diseases and allergic diseases. T cells can produce cytokines and chemokines in anti infection immunity, especially in anti tuberculosis infection Early immunity may play an important role. The delta gamma delta T cells according to the chain V region can be divided into 8 subgroups (V Delta V delta 1 to 8), normal people think that V delta 2 subgroup. Studies have shown that the proportion of V delta 1 and V delta 2 subsets changed obviously in some patients with gamma delta T cells. The former research in our laboratory was also observed in the same phenomenon. Scientific problems of our concern is: how healthy adults and patients with tuberculosis of gamma delta T cells V delta 1-8 subsets is how, if there are significant differences in healthy adults and patients with tuberculosis in particular? V 8 1 subsets and V delta 2 subsets.
Objective: changes in the peripheral blood of V delta 1-8 subsets on the distribution of peripheral blood of healthy adults in the V delta 1-8 subsets distribution and tuberculosis patients, through the contrast research of gamma delta T cells V delta 1-8 subsets, especially V delta 1 subgroup and V subgroup 8 2 in may play a different role in tuberculosis infection.
Methods: a certain number of healthy adults and patients with tuberculosis in fasting venous blood anticoagulant heparin tube in tube and incomplete standby, take 1640 medium mixed with human lymphocytes by density gradient centrifugation method for peripheral blood mononuclear cells. After washing with phosphate buffer, adjusting the cell concentration of 5 * * 106/ml1 107/ml.
Flow cytometry method of the cell suspension were added to the anti CD3-FITC, anti CD3-APC, anti V 1-PE, anti human gamma delta -TC, Delta 1-FITC, anti V, anti V 2-PE surface after staining, were detected by flow cytometry. Data were analyzed by Cell Quest software, calculate V Delta 1+, Delta 2+ V cells and V delta 1 /V delta 2 (V 8 3 ~ 8+) cells in their respective gamma delta T cells percentage.
Fluorescence quantitative RT PCR assay the cell suspension with Trizol lysis after extraction of RNA and ultra trace tester nucleic acid protein identification of RNA quality, qualified by standard samples in PCR instrument on cDNA RevertAid First Strand RT cDNASynthesis Kit kit, then cDNA SYBR Green Reagents according to the standard adding fluorescent reagent and in real time fluorescent quantitative PCR reaction, the reaction to the reference gene glyceraldehyde -3- phosphate dehydrogenase. The data will be 1-8 cycles and the difference delta gene reference after 2- conversion is obtained after the relative expression of each gene, and calculate the mean and standard deviation.
Results: 1.36 cases of healthy adults, flow cytometry, the majority of gamma delta T cells subsets of 8 2 accounted for the highest proportion of V Delta; detection by real time fluorescent quantitative RT-PCR 1-8 gene, delta 2 gene relative expression quantity was 0.0955 + 0.006, the total gamma delta T cells the expression of other genes by 57.96%. the relative expression from the bottom, followed by 3 Delta, delta delta delta 1, 7, 8, 4, 5, 6..
Among the 2.10 cases of tuberculosis patients were detected with flow cytometry, the majority of gamma delta T cells mainly in the delta 1 subsets; delta detection by real time fluorescent quantitative RT-PCR 1-8 gene, delta 1 gene relative expression quantity was 0.01723 + 0.0001, the total gamma delta T cells expression of 46.60%. delta was 2 relative gene expression for 0.0042 + 0.0007, only the total gamma delta T cells expression of 11.44%. gene in other relative expression from the bottom, followed by 5 Delta, delta delta delta 6, 4, 7, 3, 8..
3. statistical validation correlation coefficient, healthy adults group V delta 1 group, V delta 2 group, V delta 1 /V delta 2 - (V Delta 3-8+) group R2 between 0.941 to 0.985, TB group V delta group 1, V delta 2 group, V delta 3-8 group R2 0.967 to 0.967, (P0.05).
Conclusion: 1.. Flow cytometry and real-time quantitative PCR detection showed that the proportion of V delta 2 subgroup was the highest in 36 healthy adults (the total gamma delta T cells 57.96%). The proportion of other subgroups was from 3 to 3, delta 1, delta 7, delta 8, delta 4, delta 5, Delta 6..
2., compared with healthy adults, 10 cases of tuberculosis patients were mainly composed of delta 1 T subgroup (total gamma delta T cells 46.60%), and the proportion of other subgroups was from 5 to 6, delta 2, delta 4, delta 7, delta 3, Delta 8..
3., there was a strong positive linear correlation between flow cytometry and real-time quantitative PCR detection. Statistically, the subgroups of healthy adults and tuberculosis patients were statistically verified. The correlation coefficient R2 was between 0.941 and 0.990.
4., the V delta 2 cell subsets of tuberculosis patients were significantly reduced compared with healthy adults, while the V delta 1 cell subsets increased. It is concluded that the V delta 2 cell subgroup was consumed in TB infection.

【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R52

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相關(guān)期刊論文 前2條

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2 趙溫利,田志剛,李柏青;γδT細(xì)胞與腫瘤免疫[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));1997年03期

本文編號(hào)：1537122