调节性γδT细胞的体外诱导及体外扩增的γδT细胞趋化机理的初步研究

发布时间：2018-03-20 14:42

本文选题：调节性T细胞　切入点：γδT细胞　出处：《中国协和医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 第一部分小鼠调节性γ6T细胞的初步研究调节性T细胞(Regulatory T cells, Tregs)是T细胞的一个亚群,能通过影响其它免疫细胞的活性来发挥抑制免疫应答的功能。近年来,调节性T细胞成为免疫学领域的一个研究热点：经典的CD4+CD25+TCRαβTreg的功能已经基本明了,CD4+CD25- T细胞在体外经过anti-TCR/CD3e的刺激和TGF-β的诱导可以转变为CD4+CD25+Treg,并且高表达Treg的特异性标记物Foxp3。γδT细胞虽然只占总T细胞的一小部分,但其独特的抗原识别模式使得γδT细胞在免疫系统中发挥着重要的作用。近年来,许多研究结果表明γδT细胞具有免疫调节功能。我们感兴趣的是小鼠的γδT细胞是否也具有免疫调节表型的亚群,以及是否可以在体外经过anti-TCR的刺激和TGF-p的诱导而产生具有免疫调节功能的γδT细胞。我们以小鼠脾脏的T细胞为研究体系,利用流式染色和RT-PCR方法检测γδT细胞Foxp3的表达情况。结果显示,新鲜分离的小鼠脾脏γδT细胞并不表达Foxp3。然而,当脾脏T细胞在固相化anti-TCRγδ抗体刺激下培养,并添加TGF-β(浓度为5ng/ml)诱导5-8天后,Realtime PCR和免疫荧光染色结果显示,TGF-p诱导组的γδT细胞出现Foxp3的表达,与CD25表达相一致,而未诱导组的γδT细胞基本无Foxp3的表达。此外,相对于诱导组的TCRγδ+CD25-T细胞,诱导组的TCRγδ+CD25+T细胞不仅表达Foxp3,还高表达免疫调节相关分子TGF-p和GITR。随后,我们通过体外功能实验证明诱导组的γδT细胞能够明显抑制anti-CD3抗体对初始T细胞的活化和增殖作用。综上所述,小鼠γδT细胞在经过固相化anti-TCRγδ刺激和TGF-p诱导后可以产生具有免疫调节表型和功能的细胞。第二部分肿瘤过继免疫治疗中γδT细胞趋化机理的初步研究 γδT细胞是一类不同于αβT细胞的特殊细胞。近年的研究表明,无论是体内还是体外,活化的γδT细胞都具有很强的杀伤各种实体肿瘤和血液肿瘤的活性。因此γδT细胞被认为是用于肿瘤过继免疫治疗很有前景的细胞。然而γδT细胞在体内是否能够有效分布于肿瘤组织是其发挥抗肿瘤作用的前提。本实验室建立了一套固相化anti-TCRγδ抗体体外扩增培养γδT细胞的方法,所获γδT细胞制剂在体外具有很强的杀伤肿瘤细胞的活性。为了考察γδT细胞制剂在体内,特别是肿瘤组织的分布规律,我们首先分析了培养后γδT细胞表面趋化因子受体的表达情况,随后以结直肠癌为研究对象,分析了结直肠癌细胞系和结直肠癌组织标本中趋化因子的表达情况,最后利用趋化实验和趋化封闭实验考察了结直肠癌组织对γδT细胞的趋化作用。 γδT细胞制剂制备于固相化抗体体外扩增的人外周血单个核细胞,γδT细胞在扩增前后趋化因子受体的表达情况采用免疫荧光染色和流式细胞术进行分析。结果显示CCR5和CXCR3是体外扩增的Vδ1T细胞和Vδ2T细胞表面主要的趋化因子受体。用RT-PCR法检测4种人结直肠癌细胞系和10例人结直肠癌组织标本(每例均包括肿瘤组织和瘤旁组织)趋化因子的表达情况。结果表明,不同的人结直肠癌细胞系能够表达不同的趋化因子；人结直肠癌组织标本中肿瘤组织和瘤旁组织也存在多种趋化因子的表达,其中均包括CCR5和CXCR3的配体分子。因此,我们以两种结肠癌细胞系(HT-29和HR8348)为代表,进行了肿瘤细胞对γδT细胞的趋化实验,并利用CCR5和CXCR3封闭抗体考察了两种趋化因子受体在γδT细胞趋化作用中的贡献。结果显示经过体外扩增后的γδT细胞具有向肿瘤细胞迁移的能力,且趋化作用可以部分被CCR5和CXCR3抗体封闭。我们的研究结果表明,体外扩增的γδT细胞具有分布于结直肠癌组织的能力,该能力为其进一步在肿瘤局部发挥杀伤作用奠定了基础。
[Abstract]:Preliminary study on regulatory gamma 6T cells in the first part of mice
Regulatory T cells (Regulatory T cells, Tregs) is a subgroup of T cells, can play the function of suppressing the immune response by affecting the activity of other immune cells. In recent years, regulatory T cells become a hot research topic in the field of immunology: classic CD4+CD25+TCR alpha beta Treg function has been basically clear. CD4+CD25- T cells in vitro induced by anti-TCR/CD3e stimulation and TGF- beta can be changed into CD4+CD25+Treg, and high expression of cell specific markers Foxp3. gamma delta T Treg account for only a small part of the total T cells, but its unique antigen recognition pattern makes gammadelta T cells play an important role in the immune system in recent years, many research results show that gamma delta T cells have immune regulatory function. We are interested in murine gammadelta T cells also have immunomodulatory phenotype subsets, and whether you can in vitro by By anti-TCR stimulation and TGF-p and have the immunoregulatory function of gamma delta T cells. We use mouse spleen T cells as the research system, expressed by flow cytometry staining and RT-PCR Foxp3 method for the detection of gamma delta T cells. The results showed that freshly isolated spleen cells did not express T gamma delta Foxp3. however, when the T cells in the spleen of anti-TCR gammadelta antibody stimulation in solid culture, and add the TGF- beta (concentration of 5ng/ml) after 5-8 days of induction, Realtime PCR and immunofluorescence staining showed that TGF-p induced group of gamma delta T cells Foxp3 expression, consistent with the expression of CD25, expression and non induced group gamma delta T cells is essentially free of Foxp3. In addition, compared with the TCR gamma delta +CD25-T cell group, induction group TCR gamma delta +CD25+T cells not only express Foxp3, but also the high expression of immune related molecules TGF-p and GITR. then we can pass the test body The induction of gamma delta T cells in the induction group can significantly inhibit the activation and proliferation of anti-CD3 antibody on the initial T cells. In conclusion, the gamma delta T cells can produce immunoregulatory phenotype and function cells after solid anti-TCR anti-TCR delta stimulation and TGF-p induction.
Preliminary study on the chemotactic mechanism of gamma delta T cells in the second part of the tumor adoptive immunotherapy
Gamma delta T cells were different from alpha beta T cells specific cells. Recent studies have shown that either in vivo or in vitro, activation of gamma delta T cells have strong killing a variety of solid tumors and hematological tumor activity. Therefore gamma delta T cells were considered for tumor adoptive immunotherapy is promising cells. However, gamma delta T cells in vivo is effectively distributed in tumor tissue is the prerequisite for the antitumor effect. The laboratory has established a set of immobilized anti-TCR antibody in vitro culture method of gamma delta gamma delta T cells, the gamma delta T cell preparation has strong killing tumor cells the activity in vitro. In order to investigate the preparation of gamma Delta T cells in vivo, especially the distribution of tumor tissue, we first analyzed the expression of chemokine receptors on the surface of T cells after incubation, followed by colorectal cancer as the research object, analysis The expression of chemokines in colorectal cancer cell lines and colorectal cancer tissues was detected. Finally, chemotaxis and chemotactic blocking experiments were used to investigate the chemotactic effect of colorectal cancer tissues on T cells.
Gamma delta T cells in preparation of immobilized antibody in vitro amplification of human peripheral blood mononuclear cells, T cells in the amplification and expression of chemokine receptors was analyzed by immunofluorescence staining and flow cytometry. The results showed that CCR5 and CXCR3 are amplified in vitro V Delta 1T cells and surface V the main Delta 2T cell chemokine receptor. The detection of 4 human colorectal cancer cell lines and 10 cases of human colorectal carcinoma tissues by RT-PCR method (each case including tumor tissues and adjacent tissues) expression of chemokines. The results showed that different human colorectal cancer cell lines expressed different chemotaxis factor; human colorectal cancer tissues in tumor tissues and adjacent tissues of various chemokine ligand molecules, which are including CCR5 and CXCR3. Therefore, we use two kinds of colon cancer cell lines (HT-29 and HR8348) as the representative of swelling The trend of tumor cells of gamma delta T cells, and the use of CCR5 and CXCR3 blocking antibodies examined two chemokine receptors in chemotaxis contribution in gamma delta T cells. The results showed that after in vitro expansion of gamma delta T cells have the ability to migrate to tumor cells, and chemotaxis can be part of CCR5 and CXCR3 antibodies closed. Our results showed that in vitro amplification of gamma delta T cells have the ability of distribution in colorectal cancer tissues, the ability for further play in local tumor killing effect of the foundation.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【相似文献】