结核病人与健康成人外周血γδT细胞Vδ1-8亚群分布的差异

发布时间：2018-02-26 07:38

本文关键词： γδT细胞 Vδ亚群结核病人 RTPCR 流式细胞仪　出处：《蚌埠医学院》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景：结核病是严重危害人类健康的常见感染性疾病之一，但关于机体抗结核感染的免疫机制尚未完全阐明。以往研究认为，αβ T细胞中CD4+和CD8+T细胞，以及巨噬细胞在抗结核免疫中发挥关键性作用。近年来很多研究结果证实γδ T细胞在抗结核杆菌感染免疫中也发挥重要作用。γδ T细胞是与αβ T细胞有不同特征的独特T细胞，许多研究表明γδ T细胞在抗感染免疫、抗肿瘤免疫中发挥重要作用，同时γδ T细胞在调节特异性免疫应答、自身免疫性疾病和变态反应性疾病的发生均有重要的参与作用。γδ T细胞可产生多种细胞因子和趋化因子，在抗感染免疫，特别是在抗结核杆菌感染的早期免疫中可能发挥重要作用。γδ T细胞根据δ链V区不同可分为8个亚群（Vδ1至Vδ8），正常人以为Vδ2亚群为主。有研究显示，在某些疾病患者γδ T细胞中Vδ1和Vδ2亚群的比例明显改变。本研究室的前期研究中也观察到同样现象。我们关注的科学问题是：健康成年人与结核病人γδ T细胞的Vδ1-8各亚群分布情况究竟是怎样的，健康成年人与结核病人是否有明显差异？尤其是Vδ1亚群和Vδ2亚群。目的：调查健康成人外周血中Vδ1-8各亚群的分布情况和结核病人外周血中Vδ1-8各亚群的分布改变，通过研究对比，探讨γδ T细胞Vδ1-8各亚群，特别是Vδ1亚群和Vδ2亚群在结核感染中可能发挥的不同作用。方法：抽取一定数量健康成人和结核病人空腹静脉血于肝素钠抗凝管中备用，取试管与不完全1640培养液混匀，用人淋巴细胞分离液经密度梯度离心法获取外周血单个核细胞。用磷酸盐缓冲液洗涤后，调整细胞浓度5×106/ml－1×107/ml。流式细胞仪检测法取上述细胞悬液，分别加入抗人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，用流式细胞仪检测。数据分析采用Cell Quest软件，计算Vδ1+，Vδ2+细胞以及Vδ1－/Vδ2－(即Vδ3～8+)细胞各自在γδ T细胞中的百分比。荧光定量RT－PCR检测法取上述细胞悬液用Trizol裂解后提取RNA并用超微量核酸蛋白检测仪鉴定RNA质量，，合格者用RevertAid First Strand cDNASynthesis Kit试剂盒按标准加样后在PCR仪上逆转录得到cDNA，再将cDNA按标准加入SYBR Green Reagents荧光试剂，放入实时荧光定量PCR仪反应，反应参照基因为甘油醛-3-磷酸脱氢酶。所得数据将δ1-8基因循环数与内参的差值经过2-换算后得到各基因相对表达量，并计算均数和标准差。结果：1.36例健康成年人中，流式细胞仪检测，大多数人γδ T细胞δ2亚群所占比例最高；实时荧光定量RT-PCR法检测Vδ1-8基因，δ2基因相对表达量为0.0955±0.006，占总γδ T细胞表达量的57.96%。其他基因按相对表达量由多到少排列，依次为δ3、δ1、δ7、δ8、δ4、δ5、δ6。 2.10例结核病人当中，流式细胞仪检测，大多数人γδ T细胞主要以δ1亚群为主；实时荧光定量RT-PCR法检测δ1-8基因，δ1基因相对表达量为0.01723±0.0001，占总γδ T细胞表达量的46.60%。δ2基因相对表达量为0.0042±0.0007，仅占总γδ T细胞表达量的11.44%。其他基因按相对表达量由多到少排列，依次为δ5、δ6、δ4、δ7、δ3、δ8。 3.统计学验证相关系数，健康成年人组的Vδ1组，Vδ2组，Vδ1－/Vδ2－(即Vδ3-8+)组R2在0.941到0.985间，结核病人组的Vδ1组，Vδ2组，Vδ3-8组R2在0.967到0.990间，(P0.05)。结论：1.流式细胞术和实时定量PCR技术检测发现，36例健康成年人γδ T细胞亚群中，以Vδ2亚群比例最高（占总γδ T细胞57.96%），其他亚群的比例由多到少依次为δ3、δ1、δ7、δ8、δ4、δ5、δ6。 2.与健康成年人不同，10例结核病人的γδ T细胞主要以δ1亚群为主（占总γδT细胞46.60%），其他亚群的比例由多到少依次为δ5、δ6、δ2、δ4、δ7、δ3、δ8。 3.流式细胞仪检测法与实时定量PCR检测法呈较强的线性正相关关系，统计学验证健康成年人组和结核病人组不同亚群，相关系数R2在0.941到0.990间。 4.结核病人Vδ2细胞亚群较健康成年人明显减少，而Vδ1细胞亚群相对增高。推断该Vδ2细胞亚群在结核感染中被消耗所导致。
[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.

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R52

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