【摘要】：鈣調磷酸酶抑制劑對小鼠Thl7細胞分化增殖的影響及其機制探討 目的 對許多終末期實體臟器疾病來說，器官移植是-個有效治療手段。但是，盡管目前免疫抑制治療取得了很大進展，移植排斥反應依然是影響受體長期存活的重要因素之-。th17細胞是2005年新發(fā)現(xiàn)的效應CD4_T細胞亞群，產生致炎細胞因子IL-17AF、IL6等，主要介導炎癥反應、自身免疫病等的發(fā)生發(fā)展。文獻報道，除th1細胞外，th17細胞也參與了移植排斥反應的進程；當th1細胞反應被抑制時，th17細胞可能發(fā)揮了更重要的作用。深入了解影響各亞群細胞分化增殖的因素有利于防治移植排斥反應。而鈣調磷酸酶抑制劑作為目前移植臨床最常用的免疫抑制劑，其與th17細胞的關系，迄今為止尚未闡明。因此，，本課題通過人工建立th17極化環(huán)境培育出th17細胞，并建立小鼠頸部心臟移植模型，研究鈣調磷酸酶抑制劑對小鼠th17細胞分化增殖的影響并探討其機制；此外，通過體內實驗研究th17細胞相關因子IL-17在小鼠心臟移植排斥反應中的表達及其意義。 方法 小鼠脾臟初始CD4~+CD25 T淋巴細胞使用抗CD3抗體及抗CD28抗體活化，1IGFBl及IL6等細胞因子誘導，促使其向th17細胞方向分化；施加不同劑量的鈣調磷酸酶抑制劑如他克莫司或環(huán)孢素A進行干予頁。使用流式細胞儀檢測各組CD4’th17細胞亞群純度，使用R_r_PCR及WesternBl0t檢測各組Il_17mRNA相對量。建立小鼠頸部異位心臟移植模型，實驗動物隨機分組，分別為同系移植組，異系移植組，CA組及FK506組，觀察各組供心存活時間；應用RT．PCR檢測移植心臟IL-17mRNA、IFNv mRNA在移植術后2、4、6、8d的動態(tài)表達水平；病理切片了解發(fā)生排斥反應與否。 結果 本課題通過使用鈣調磷酸酶抑制劑(他克莫司與環(huán)孢素A)，分別在體外環(huán)境和體內環(huán)境中進行干予頁，發(fā)現(xiàn)th17細胞的分化增殖受到明顯抑制；同時，小鼠心臟的急性排斥反應也得到了有效控制：鈣調磷酸酶抑制劑抑制小鼠th17細胞亞群的分化增殖，且呈劑量依賴性。各組問差異有顯著性(P0．05)。此外，構建小鼠頸部異位心臟移植的急性排斥反應模型，觀察th1、th17細胞因子在急性排斥反應不同階段的表達情況。研究結果顯示，Il，17參與了急性排斥反應的進程。在異系移植組(急性排斥反應組)中，術后第2天即可檢測到ILl7 mRNA的表達，其表達量在術后第4d達丑高峰，隨后逐漸下降，在術后第6d無表達。而th1細胞的主要細胞因子IFNv mRNA的表達則晚于IL-17 mRNA。這從側面說明th17細胞和th1細胞在急性排斥反應的不同階段分別發(fā)揮著重要作用。 結論 th17細胞在移植排斥反應的進程中起著重要作用，對IL-17的檢測可以作為急性排斥反應早期診斷的予頁見性指標。而鈣調磷酸酶抑制劑除了可以抑制th1細胞外，對th17細胞的分化增殖和IL-17的釋放也有著強大的抑制作用。以上研究發(fā)現(xiàn)，均有利于我們進-步了解移植排斥反應的進程和指導臨床防治。本課題探討了鈣調磷酸酶抑制劑對小鼠th17細胞的影響：通過體外實驗及體內實驗發(fā)現(xiàn)，在鈣調磷酸酶抑制劑存在的環(huán)境下，th17細胞亞群分化增殖明顯減少；IL-17mRNA表達降低，且呈齊【量依賴性。這種現(xiàn)象需要新的機理進-步解釋。經廣泛閱讀文獻發(fā)現(xiàn)：Gomez—R0drigucz等在對T細胞受體信號通路及ITK蛋白的研究中提出：th17細胞內il基因的表達除了受RORm和S1_AT3轉導的細胞因子刺激外，還要接受TCR信號通路鈣調磷酸酶介導信號的刺激。th17細胞細胞因子IL-17A的表達與Ca2+內流和NFAR激活有關；反之，1ICR信號通路刺激減少或鈣調磷酸酶受抑制優(yōu)先減少了IL-17A的表達。由此推理得出結論，鈣調磷酸酶抑制劑抑制th17細胞胞漿內的鈣調磷酸酶，從而阻止NFA_rc的脫磷酸化和核內轉移過程，進-步抑制il基因的轉錄活性，抑制Il，17A產生并減輕炎癥進程，從另-途徑抑制了排斥反應。鈣調磷酸酶抑制劑可以抑制th17細胞亞群的分化增殖。本課題進-步闡明了鈣調磷酸酶抑制劑發(fā)揮免疫抑制作用的機制，為臨床上科學應用鈣調磷酸酶抑制劑提供實驗依據(jù)和理論基礎。
[Abstract]:Effects of calcineurin inhibitors on differentiation and proliferation of mouse Thl7 cells and its mechanism
objective
Organ transplantation is an effective treatment for many end-stage organ diseases. However, despite the great progress made in immunosuppressive therapy, transplant rejection is still an important factor affecting the long-term survival of recipients - - Th17 cells are the newly discovered effector CD4_T cell subsets, producing inflammatory cytokine IL-17AF, in 2005. In addition to Th1 cells, Th17 cells also participate in the process of graft rejection. When Th1 cell reaction is inhibited, Th17 cells may play a more important role. Understanding the factors affecting the differentiation and proliferation of each subgroup of cells is conducive to the prevention and treatment of transplantation. The relationship between calmodulin phosphatase inhibitors and Th17 cells has not been elucidated so far. Therefore, Th17 cells were cultured in the polarized environment of th17, and a mouse model of cervical heart transplantation was established to study the effects of calmodulin phosphatase inhibitors on Th17 cells in mice. In addition, the expression and significance of interleukin-17 (IL-17), a cytokine related to Th17 cells, were studied in vivo.
Method
Mouse spleen initial CD4~+CD25 T lymphocytes were activated by anti-CD3 antibody and anti-CD28 antibody, and induced to differentiate into Th17 cells by cytokines such as 1IGFBl and IL6. Different doses of calmodulin inhibitors such as tacrolimus or cyclosporine A were applied to dry-feed the lymphocytes. Purity was measured by R_r_PCR and Western Bl0t. A mouse model of heterotopic heart transplantation was established. The experimental animals were randomly divided into homologous transplantation group, allogeneic transplantation group, CA group and FK506 group to observe the survival time of donor hearts; IL-17 mRNA was detected by RT.PCR and IFNv mRNA was detected at 2,4,6,8 days after transplantation. The dynamic expression level and pathological section were used to know whether rejection occurred.
Result
Calmodulin inhibitors (tacrolimus and cyclosporine A) were used to dry the leaves of Th17 cells in vitro and in vivo, respectively. It was found that the differentiation and proliferation of Th17 cells were significantly inhibited. At the same time, the acute rejection of mouse heart was effectively controlled: Calmodulin inhibitors inhibited Th17 cell subsets in mice. In addition, the acute rejection model of mouse cervical heterotopic heart transplantation was established to observe the expression of Th1 and Th17 cytokines in different stages of acute rejection. In group A (acute rejection group), the expression of IL-7 mRNA was detected on the 2nd day after operation. The expression of IL-7 mRNA reached an ugly peak on the 4th day after operation, then decreased gradually, and was not expressed on the 6th day after operation. The expression of IFNv mRNA in Th1 cells was later than that in IL-17 mRNA. At the same stage, they played an important role respectively.
conclusion
Th17 cells play an important role in the process of transplantation rejection, and the detection of IL-17 can be used as a predictive marker for early diagnosis of acute rejection. Calmodulin phosphatase inhibitors can inhibit the differentiation and proliferation of Th17 cells and the release of IL-17 in addition to Th1 cells. This study explored the effects of calmodulin phosphatase inhibitors on Th17 cells in vitro and in vivo. It was found that in the presence of calmodulin phosphatase inhibitors, the differentiation and proliferation of Th17 cell subsets were significantly reduced. This phenomenon requires a new mechanism to be further explained. After extensive reading of the literature, Gomez-R0 drigucz et al. have proposed that the expression of IL gene in Th17 cells is stimulated by RORm and S1_AT3 transduction cytokines in addition to T cell receptor signaling pathway and ITK protein. The expression of cytokine IL-17A in Th17 cells is related to Ca2+ influx and NFAR activation, whereas the decrease of stimulation of 1ICR signaling pathway or inhibition of calcineurin preferentially reduces the expression of IL-17A. Calmodulin phosphatase inhibitors can inhibit the differentiation and proliferation of Th17 cell subsets. In this study, we further clarified that calmodulin phosphatase inhibitors play an immunosuppressive role. The mechanism of action will provide experimental evidence and theoretical basis for clinical application of calcineurin inhibitors.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R392;R654.2

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相關期刊論文 前1條

1 朱曉星;楊康;吳蔚;;近交系小鼠不同品系頸部心臟移植[J];現(xiàn)代生物醫(yī)學進展;2008年01期

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