【摘要】：目的:本文主要探究人趨化因子CCL17和CCL22對淋巴細(xì)胞的趨化能力及抗腫瘤功能,從而把CCL17和CCL22與殺傷性免疫細(xì)胞聯(lián)系起來,使具有非限制性腫瘤殺傷的CIK細(xì)胞高表達(dá)CCL17和CCL22的共同配體CCR4,從而使其能被趨化到腫瘤周圍進(jìn)行免疫殺傷。方法:1、人趨化因子CCL17和CCL22對CD4及CD8兩種T淋巴細(xì)胞亞群的趨化能力比較研究本節(jié)我們重點(diǎn)研究了CCL17和CCL22對CD4和CD8兩種T細(xì)胞趨化能力上的差別以及對不同T細(xì)胞亞群表面CCR4分子內(nèi)化能力的影響。我們首先運(yùn)用流式細(xì)胞儀分析了健康人外周血PBMC中CCR4+T細(xì)胞的分布情況,接著我們利用Mo Flo分選流式細(xì)胞儀分選出CD4和CD8T淋巴細(xì)胞,進(jìn)行細(xì)胞趨化實(shí)驗(yàn),利用Transwell板,我們檢測了不同濃度下,兩種趨化因子對CD4和CD8T細(xì)胞的趨化效果,我們進(jìn)一步研究了CCL17和CCL22對CD4和CD8細(xì)胞的CCR4受體內(nèi)化情況。趨化因子結(jié)合細(xì)胞表面受體后會(huì)引起受體分子脫敏而發(fā)生內(nèi)化現(xiàn)象,受體內(nèi)化的程度和比例決定著受體信號通路的活化。2、CCL17和CCL22對CIK和DC-CIK的受體表達(dá)影響本節(jié)實(shí)驗(yàn)中我們首先檢測了CCR4在CIK和DC-CIK細(xì)胞上的表達(dá)情況,并且做了CCL17和CCL22對CIK和DC-CIK細(xì)胞的趨化實(shí)驗(yàn),流式分析DC-CIK和CIK表達(dá)的CCR4,推測是DC分泌的CCL17和CCL22致使DC-CIK表面CCR4表達(dá)升高,然后測定DC-CIK和CIK細(xì)胞的上清中CCL17和CCL22的含量,最后用一定濃度的兩種趨化因子作用CIK細(xì)胞一段時(shí)間,刺激CIK細(xì)胞上面表達(dá)CCR4。3、轉(zhuǎn)染CCR4的CIK細(xì)胞的趨化功能以及對腫瘤的殺傷作用研究本節(jié)我們主要做的是把基因CCR4轉(zhuǎn)染到CIK細(xì)胞上,使CIK細(xì)胞高表達(dá)CCR4基因,進(jìn)而比較轉(zhuǎn)染CCR4的CIK細(xì)胞和未轉(zhuǎn)染的CIK細(xì)胞的趨化作用和對腫瘤殺傷作用。結(jié)果:1、作用于同一受體分子CCR4,濃度為100ng/ml的兩種趨化因子趨化CD4和CD8的能力均強(qiáng)于10ng/ml,表明在一定的濃度范圍內(nèi),濃度越高的趨化因子對細(xì)胞的趨化能力越強(qiáng)。在相同趨化時(shí)間內(nèi),CCL22對CD4和CD8兩種T細(xì)胞亞群的趨化能力強(qiáng)于CCL17,CCL17和CCL22趨化CD4細(xì)胞通過Transwell板的細(xì)胞數(shù)均多于CD8細(xì)胞,但CCL17對兩種T細(xì)胞亞群趨化能力的差異性要小于CCL22。CCR4受體內(nèi)化實(shí)驗(yàn)證實(shí),相同時(shí)間下CCL22比CCL17更容易促使CCR4受體發(fā)生內(nèi)化(P0.05),對于CD4和CD8兩種T細(xì)胞亞群,CCL22和CCL17作用后受體CCR4后CD4細(xì)胞的內(nèi)化作用要強(qiáng)于CD8細(xì)胞。2、CIK細(xì)胞在14天培養(yǎng)過程中CCR4的表達(dá)先增多后減少,第四天左右最高,培養(yǎng)時(shí)間越長CCR4表達(dá)量越來越低,直到最后回收CIK細(xì)胞時(shí)CCR4幾乎不再表達(dá)。DC-CIK細(xì)胞隨著第十天DC細(xì)胞的加入直到第十四天回收細(xì)胞,這期間CCR4的表達(dá)量不僅沒有降低反而升高了,原因是由于DC分泌趨化因子CCL22和CCL17導(dǎo)致的CCR4表達(dá)增高。在進(jìn)行細(xì)胞免疫治療時(shí)可以加入適當(dāng)濃度的CCL17和CCL22,促進(jìn)細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞CIK表面配體CCR4的表達(dá),而腫瘤細(xì)胞周圍環(huán)境中往往會(huì)分泌這兩種趨化因子,免疫細(xì)胞表面表達(dá)的CCR4越多,腫瘤微環(huán)境就可以趨化更多的表達(dá)CCR4配體的免疫細(xì)胞進(jìn)行腫瘤殺傷。3、把基因CCR4轉(zhuǎn)染到CIK細(xì)胞上,使CIK細(xì)胞高表達(dá)CCR4基因,進(jìn)而轉(zhuǎn)染CCR4的CIK細(xì)胞比未轉(zhuǎn)染的CIK細(xì)胞有更強(qiáng)的趨化作用和更強(qiáng)的殺傷作用。對腫瘤的殺傷有重大意義。由于許多腫瘤微環(huán)境周圍被證明高表達(dá)CCL17和CCL22兩種趨化因子,所以腫瘤細(xì)胞周圍的液體環(huán)境可以趨化更多的免疫細(xì)胞進(jìn)行殺傷腫瘤,這將對腫瘤的殺傷有重大意義。結(jié)論:一定的濃度范圍內(nèi),濃度越高的趨化因子對細(xì)胞的趨化能力越強(qiáng)。CCL22比CCL17更能促進(jìn)CCR4+T細(xì)胞的趨化運(yùn)動(dòng)和發(fā)生受體內(nèi)化現(xiàn)象,對于CD4和CD8細(xì)胞而言,CCL22和CCL17對CD4細(xì)胞的趨化效能和內(nèi)化能力強(qiáng)于CD8細(xì)胞。免疫細(xì)胞表面表達(dá)的CCR4越多,腫瘤微環(huán)境就可以趨化更多的表達(dá)CCR4配體的免疫細(xì)胞進(jìn)行腫瘤殺傷。我們的研究就是把CCL17和CCL22與殺傷性免疫細(xì)胞聯(lián)系起來,讓這些具有非限制性腫瘤殺傷的CIK細(xì)胞高表達(dá)CCL17和CCL22的共同配體CCR4,從而使其能被趨化到腫瘤周圍進(jìn)行免疫殺傷而不是像高表達(dá)CCR4的Treg細(xì)胞那樣抑制腫瘤殺傷。
[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.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R730.51

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本文編號：2136739