人趋化因子CCL17和CCL22对淋巴细胞抗肿瘤功能的影响研究
发布时间：2018-07-21 20:18
【摘要】：目的:本文主要探究人趋化因子CCL17和CCL22对淋巴细胞的趋化能力及抗肿瘤功能,从而把CCL17和CCL22与杀伤性免疫细胞联系起来,使具有非限制性肿瘤杀伤的CIK细胞高表达CCL17和CCL22的共同配体CCR4,从而使其能被趋化到肿瘤周围进行免疫杀伤。方法:1、人趋化因子CCL17和CCL22对CD4及CD8两种T淋巴细胞亚群的趋化能力比较研究本节我们重点研究了CCL17和CCL22对CD4和CD8两种T细胞趋化能力上的差别以及对不同T细胞亚群表面CCR4分子内化能力的影响。我们首先运用流式细胞仪分析了健康人外周血PBMC中CCR4+T细胞的分布情况,接着我们利用Mo Flo分选流式细胞仪分选出CD4和CD8T淋巴细胞,进行细胞趋化实验,利用Transwell板,我们检测了不同浓度下,两种趋化因子对CD4和CD8T细胞的趋化效果,我们进一步研究了CCL17和CCL22对CD4和CD8细胞的CCR4受体内化情况。趋化因子结合细胞表面受体后会引起受体分子脱敏而发生内化现象,受体内化的程度和比例决定着受体信号通路的活化。2、CCL17和CCL22对CIK和DC-CIK的受体表达影响本节实验中我们首先检测了CCR4在CIK和DC-CIK细胞上的表达情况,并且做了CCL17和CCL22对CIK和DC-CIK细胞的趋化实验,流式分析DC-CIK和CIK表达的CCR4,推测是DC分泌的CCL17和CCL22致使DC-CIK表面CCR4表达升高,然后测定DC-CIK和CIK细胞的上清中CCL17和CCL22的含量,最后用一定浓度的两种趋化因子作用CIK细胞一段时间,刺激CIK细胞上面表达CCR4。3、转染CCR4的CIK细胞的趋化功能以及对肿瘤的杀伤作用研究本节我们主要做的是把基因CCR4转染到CIK细胞上,使CIK细胞高表达CCR4基因,进而比较转染CCR4的CIK细胞和未转染的CIK细胞的趋化作用和对肿瘤杀伤作用。结果:1、作用于同一受体分子CCR4,浓度为100ng/ml的两种趋化因子趋化CD4和CD8的能力均强于10ng/ml,表明在一定的浓度范围内,浓度越高的趋化因子对细胞的趋化能力越强。在相同趋化时间内,CCL22对CD4和CD8两种T细胞亚群的趋化能力强于CCL17,CCL17和CCL22趋化CD4细胞通过Transwell板的细胞数均多于CD8细胞,但CCL17对两种T细胞亚群趋化能力的差异性要小于CCL22。CCR4受体内化实验证实,相同时间下CCL22比CCL17更容易促使CCR4受体发生内化(P0.05),对于CD4和CD8两种T细胞亚群,CCL22和CCL17作用后受体CCR4后CD4细胞的内化作用要强于CD8细胞。2、CIK细胞在14天培养过程中CCR4的表达先增多后减少,第四天左右最高,培养时间越长CCR4表达量越来越低,直到最后回收CIK细胞时CCR4几乎不再表达。DC-CIK细胞随着第十天DC细胞的加入直到第十四天回收细胞,这期间CCR4的表达量不仅没有降低反而升高了,原因是由于DC分泌趋化因子CCL22和CCL17导致的CCR4表达增高。在进行细胞免疫治疗时可以加入适当浓度的CCL17和CCL22,促进细胞因子诱导的杀伤细胞CIK表面配体CCR4的表达,而肿瘤细胞周围环境中往往会分泌这两种趋化因子,免疫细胞表面表达的CCR4越多,肿瘤微环境就可以趋化更多的表达CCR4配体的免疫细胞进行肿瘤杀伤。3、把基因CCR4转染到CIK细胞上,使CIK细胞高表达CCR4基因,进而转染CCR4的CIK细胞比未转染的CIK细胞有更强的趋化作用和更强的杀伤作用。对肿瘤的杀伤有重大意义。由于许多肿瘤微环境周围被证明高表达CCL17和CCL22两种趋化因子,所以肿瘤细胞周围的液体环境可以趋化更多的免疫细胞进行杀伤肿瘤,这将对肿瘤的杀伤有重大意义。结论:一定的浓度范围内,浓度越高的趋化因子对细胞的趋化能力越强。CCL22比CCL17更能促进CCR4+T细胞的趋化运动和发生受体内化现象,对于CD4和CD8细胞而言,CCL22和CCL17对CD4细胞的趋化效能和内化能力强于CD8细胞。免疫细胞表面表达的CCR4越多,肿瘤微环境就可以趋化更多的表达CCR4配体的免疫细胞进行肿瘤杀伤。我们的研究就是把CCL17和CCL22与杀伤性免疫细胞联系起来,让这些具有非限制性肿瘤杀伤的CIK细胞高表达CCL17和CCL22的共同配体CCR4,从而使其能被趋化到肿瘤周围进行免疫杀伤而不是像高表达CCR4的Treg细胞那样抑制肿瘤杀伤。
[Abstract]:Objective: To explore the chemotaxis and anti-tumor functions of human chemokine CCL17 and CCL22, and to link CCL17 and CCL22 with killer immune cells, so that CIK cells with non restrictive tumor killing can express the common ligand CCR4 of CCL17 and CCL22, so that they can be immunized around the tumor for immunity. Methods: 1, 1, human chemotactic factor CCL17 and CCL22 comparative study on chemotaxis of two T lymphocyte subsets of CD4 and CD8. We focus on the difference between CCL17 and CCL22 on the chemotaxis of CD4 and CD8 two T cells and the effect on the internalized energy of the surface CCR4 molecules on the surface of different T cell subsets. The distribution of CCR4+T cells in the peripheral blood PBMC of healthy people was analyzed. Then we used Mo Flo separation flow cytometry to select CD4 and CD8T lymphocytes, carry out cell chemotaxis experiments and use Transwell plates. We detected the chemotaxis effect of two chemokines on CD4 and CD8T cells at different concentrations. We further studied CCL17 and CCL. 22 the internalization of CCR4 receptor in CD4 and CD8 cells. Chemokine binding to the cell surface receptor causes the internalization of receptor molecules desensitization. The degree and proportion of receptor internalization determines the activation.2 of the receptor signaling pathway. CCL17 and CCL22 are the first to detect CCR4 in CIK in the receptor expression of CIK and DC-CIK. The expression on DC-CIK cells and the chemotactic experiment of CIK and DC-CIK cells by CCL17 and CCL22. Flow analysis of DC-CIK and CIK expressed CCR4. It is presumed that CCL17 and CCL22 of DC secretion increase the expression of DC-CIK surface, and then determine the content and content in the supernatant of the cells. Finally, two species of certain concentration are used. Chemokines effect CIK cells for a period of time, stimulating the expression of CCR4.3 on the CIK cells, the chemotaxis function of CIK cells transfected with CCR4 and the killing effect on the tumor. The main thing we do is to transfect the gene CCR4 into CIK cells and make the CIK cells highly express the CCR4 gene, and then compare CIK and untransfected CIK cells to the CIK cells. Chemotaxis and tumor killing effect. Results: 1, the ability of two chemokines to chemotaxis CD4 and CD8 of the same receptor molecule CCR4, the concentration of 100ng/ml, is stronger than 10ng/ml, indicating that the chemotactic factor of higher concentration is stronger than 10ng/ml in a certain concentration range. In the same chemotactic time, CCL22 is to CD4 and CD8 two. The chemotaxis of T cell subsets is stronger than that of CCL17, and the number of CCL17 and CCL22 chemotactic CD4 cells through Transwell plate is more than CD8 cells, but the difference of CCL17 to the chemotaxis ability of two T cell subsets is less than that of CCL22.CCR4 receptor internalization experiment. 4 and CD8 two T cell subsets, the internalization of CD4 cells after CCL22 and CCL17 receptor CCR4 is stronger than CD8 cell.2, and the expression of CCR4 is increased first and then decreases in the 14 day culture process, and the longer the incubation time is, the longer the CCR4 expression is lower and lower. With the addition of DC cells for tenth days until the fourteenth day of cell recovery, the expression of CCR4 not only did not decrease but increased, due to the increase of CCR4 expression caused by DC secreting chemokine CCL22 and CCL17. In cell immunotherapy, the appropriate concentration of CCL17 and CCL22 can be added to promote cytokine induction. The expression of CCR4 on the surface of the killer cell CIK surface ligand, while the two chemokines are often secreted in the surrounding environment of the tumor cells. The more CCR4 expressed on the surface of the immune cells, the tumor microenvironment can chemotactic more CCR4 ligand immune cells to kill.3, and transfect the gene CCR4 to CIK cells, so that the CIK cells are highly expressed in CCR4. The gene, then transfected with CCR4 CIK cells, has stronger chemotaxis and stronger killing effects than untransfected CIK cells. It is of great significance to the tumor's killing. As many tumor microenvironments are shown to express the two chemokines of CCL17 and CCL22, the liquid environment around the tumor cells can chemotaxis more immune cells. In a certain concentration range, the greater the concentration of chemokine, the stronger the chemotactic capacity of cells, the more.CCL22 can promote chemotaxis and receptor internalization of CCR4+T cells than that of CCL17. For CD4 and CD8 cells, CCL22 and CCL17 have chemotaxis to CD4 cells. And internalization is stronger than CD8 cells. The more CCR4 is expressed on the surface of the immune cells, the tumor microenvironment can chemotactic more CCR4 ligand immune cells to kill the tumor. Our study is to associate CCL17 and CCL22 with killer immune cells to make these CIK cells highly expressed by non restrictive tumor cells to express CCL17 The common ligand CCR4, CCL22, can be chemotactic to the surrounding tumor to carry out immune killing rather than inhibiting tumor killing as high as CCR4 Treg cells.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.51
本文编号：2136739
[Abstract]:Objective: To explore the chemotaxis and anti-tumor functions of human chemokine CCL17 and CCL22, and to link CCL17 and CCL22 with killer immune cells, so that CIK cells with non restrictive tumor killing can express the common ligand CCR4 of CCL17 and CCL22, so that they can be immunized around the tumor for immunity. Methods: 1, 1, human chemotactic factor CCL17 and CCL22 comparative study on chemotaxis of two T lymphocyte subsets of CD4 and CD8. We focus on the difference between CCL17 and CCL22 on the chemotaxis of CD4 and CD8 two T cells and the effect on the internalized energy of the surface CCR4 molecules on the surface of different T cell subsets. The distribution of CCR4+T cells in the peripheral blood PBMC of healthy people was analyzed. Then we used Mo Flo separation flow cytometry to select CD4 and CD8T lymphocytes, carry out cell chemotaxis experiments and use Transwell plates. We detected the chemotaxis effect of two chemokines on CD4 and CD8T cells at different concentrations. We further studied CCL17 and CCL. 22 the internalization of CCR4 receptor in CD4 and CD8 cells. Chemokine binding to the cell surface receptor causes the internalization of receptor molecules desensitization. The degree and proportion of receptor internalization determines the activation.2 of the receptor signaling pathway. CCL17 and CCL22 are the first to detect CCR4 in CIK in the receptor expression of CIK and DC-CIK. The expression on DC-CIK cells and the chemotactic experiment of CIK and DC-CIK cells by CCL17 and CCL22. Flow analysis of DC-CIK and CIK expressed CCR4. It is presumed that CCL17 and CCL22 of DC secretion increase the expression of DC-CIK surface, and then determine the content and content in the supernatant of the cells. Finally, two species of certain concentration are used. Chemokines effect CIK cells for a period of time, stimulating the expression of CCR4.3 on the CIK cells, the chemotaxis function of CIK cells transfected with CCR4 and the killing effect on the tumor. The main thing we do is to transfect the gene CCR4 into CIK cells and make the CIK cells highly express the CCR4 gene, and then compare CIK and untransfected CIK cells to the CIK cells. Chemotaxis and tumor killing effect. Results: 1, the ability of two chemokines to chemotaxis CD4 and CD8 of the same receptor molecule CCR4, the concentration of 100ng/ml, is stronger than 10ng/ml, indicating that the chemotactic factor of higher concentration is stronger than 10ng/ml in a certain concentration range. In the same chemotactic time, CCL22 is to CD4 and CD8 two. The chemotaxis of T cell subsets is stronger than that of CCL17, and the number of CCL17 and CCL22 chemotactic CD4 cells through Transwell plate is more than CD8 cells, but the difference of CCL17 to the chemotaxis ability of two T cell subsets is less than that of CCL22.CCR4 receptor internalization experiment. 4 and CD8 two T cell subsets, the internalization of CD4 cells after CCL22 and CCL17 receptor CCR4 is stronger than CD8 cell.2, and the expression of CCR4 is increased first and then decreases in the 14 day culture process, and the longer the incubation time is, the longer the CCR4 expression is lower and lower. With the addition of DC cells for tenth days until the fourteenth day of cell recovery, the expression of CCR4 not only did not decrease but increased, due to the increase of CCR4 expression caused by DC secreting chemokine CCL22 and CCL17. In cell immunotherapy, the appropriate concentration of CCL17 and CCL22 can be added to promote cytokine induction. The expression of CCR4 on the surface of the killer cell CIK surface ligand, while the two chemokines are often secreted in the surrounding environment of the tumor cells. The more CCR4 expressed on the surface of the immune cells, the tumor microenvironment can chemotactic more CCR4 ligand immune cells to kill.3, and transfect the gene CCR4 to CIK cells, so that the CIK cells are highly expressed in CCR4. The gene, then transfected with CCR4 CIK cells, has stronger chemotaxis and stronger killing effects than untransfected CIK cells. It is of great significance to the tumor's killing. As many tumor microenvironments are shown to express the two chemokines of CCL17 and CCL22, the liquid environment around the tumor cells can chemotaxis more immune cells. In a certain concentration range, the greater the concentration of chemokine, the stronger the chemotactic capacity of cells, the more.CCL22 can promote chemotaxis and receptor internalization of CCR4+T cells than that of CCL17. For CD4 and CD8 cells, CCL22 and CCL17 have chemotaxis to CD4 cells. And internalization is stronger than CD8 cells. The more CCR4 is expressed on the surface of the immune cells, the tumor microenvironment can chemotactic more CCR4 ligand immune cells to kill the tumor. Our study is to associate CCL17 and CCL22 with killer immune cells to make these CIK cells highly expressed by non restrictive tumor cells to express CCL17 The common ligand CCR4, CCL22, can be chemotactic to the surrounding tumor to carry out immune killing rather than inhibiting tumor killing as high as CCR4 Treg cells.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.51
【参考文献】
相关期刊论文 前3条
1 ;Autologous cytokine-induced killer cell therapy in clinical trial phase I is safe in patients with primary hepatocellular carcinoma[J];World Journal of Gastroenterology;2004年08期
2 ;Antitumor activities of human autologous cytokineinduced killer(CIK)cells against hepatocellular carcinoma cells in vitro and in vivo[J];World Journal of Gastroenterology;2002年03期
3 于津浦,任秀宝;CIK细胞-肿瘤过继免疫治疗的新希望[J];中国肿瘤临床;2001年07期
，本文编号：2136739
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2136739.html
