钙调磷酸酶抑制剂对小鼠Th17细胞分化增殖的影响及其机制探讨
发布时间：2018-08-29 17:51
【摘要】：钙调磷酸酶抑制剂对小鼠Thl7细胞分化增殖的影响及其机制探讨 目的 对许多终末期实体脏器疾病来说，器官移植是-个有效治疗手段。但是，尽管目前免疫抑制治疗取得了很大进展，移植排斥反应依然是影响受体长期存活的重要因素之-。th17细胞是2005年新发现的效应CD4_T细胞亚群，产生致炎细胞因子IL-17AF、IL6等，主要介导炎症反应、自身免疫病等的发生发展。文献报道，除th1细胞外，th17细胞也参与了移植排斥反应的进程；当th1细胞反应被抑制时，th17细胞可能发挥了更重要的作用。深入了解影响各亚群细胞分化增殖的因素有利于防治移植排斥反应。而钙调磷酸酶抑制剂作为目前移植临床最常用的免疫抑制剂，其与th17细胞的关系，迄今为止尚未阐明。因此，，本课题通过人工建立th17极化环境培育出th17细胞，并建立小鼠颈部心脏移植模型，研究钙调磷酸酶抑制剂对小鼠th17细胞分化增殖的影响并探讨其机制；此外，通过体内实验研究th17细胞相关因子IL-17在小鼠心脏移植排斥反应中的表达及其意义。 方法 小鼠脾脏初始CD4~+CD25 T淋巴细胞使用抗CD3抗体及抗CD28抗体活化，1IGFBl及IL6等细胞因子诱导，促使其向th17细胞方向分化；施加不同剂量的钙调磷酸酶抑制剂如他克莫司或环孢素A进行干予页。使用流式细胞仪检测各组CD4’th17细胞亚群纯度，使用R_r_PCR及WesternBl0t检测各组Il_17mRNA相对量。建立小鼠颈部异位心脏移植模型，实验动物随机分组，分别为同系移植组，异系移植组，CA组及FK506组，观察各组供心存活时间；应用RT．PCR检测移植心脏IL-17mRNA、IFNv mRNA在移植术后2、4、6、8d的动态表达水平；病理切片了解发生排斥反应与否。 结果 本课题通过使用钙调磷酸酶抑制剂(他克莫司与环孢素A)，分别在体外环境和体内环境中进行干予页，发现th17细胞的分化增殖受到明显抑制；同时，小鼠心脏的急性排斥反应也得到了有效控制：钙调磷酸酶抑制剂抑制小鼠th17细胞亚群的分化增殖，且呈剂量依赖性。各组问差异有显著性(P0．05)。此外，构建小鼠颈部异位心脏移植的急性排斥反应模型，观察th1、th17细胞因子在急性排斥反应不同阶段的表达情况。研究结果显示，Il，17参与了急性排斥反应的进程。在异系移植组(急性排斥反应组)中，术后第2天即可检测到ILl7 mRNA的表达，其表达量在术后第4d达丑高峰，随后逐渐下降，在术后第6d无表达。而th1细胞的主要细胞因子IFNv mRNA的表达则晚于IL-17 mRNA。这从侧面说明th17细胞和th1细胞在急性排斥反应的不同阶段分别发挥着重要作用。 结论 th17细胞在移植排斥反应的进程中起着重要作用，对IL-17的检测可以作为急性排斥反应早期诊断的予页见性指标。而钙调磷酸酶抑制剂除了可以抑制th1细胞外，对th17细胞的分化增殖和IL-17的释放也有着强大的抑制作用。以上研究发现，均有利于我们进-步了解移植排斥反应的进程和指导临床防治。本课题探讨了钙调磷酸酶抑制剂对小鼠th17细胞的影响：通过体外实验及体内实验发现，在钙调磷酸酶抑制剂存在的环境下，th17细胞亚群分化增殖明显减少；IL-17mRNA表达降低，且呈齐【量依赖性。这种现象需要新的机理进-步解释。经广泛阅读文献发现：Gomez—R0drigucz等在对T细胞受体信号通路及ITK蛋白的研究中提出：th17细胞内il基因的表达除了受RORm和S1_AT3转导的细胞因子刺激外，还要接受TCR信号通路钙调磷酸酶介导信号的刺激。th17细胞细胞因子IL-17A的表达与Ca2+内流和NFAR激活有关；反之，1ICR信号通路刺激减少或钙调磷酸酶受抑制优先减少了IL-17A的表达。由此推理得出结论，钙调磷酸酶抑制剂抑制th17细胞胞浆内的钙调磷酸酶，从而阻止NFA_rc的脱磷酸化和核内转移过程，进-步抑制il基因的转录活性，抑制Il，17A产生并减轻炎症进程，从另-途径抑制了排斥反应。钙调磷酸酶抑制剂可以抑制th17细胞亚群的分化增殖。本课题进-步阐明了钙调磷酸酶抑制剂发挥免疫抑制作用的机制，为临床上科学应用钙调磷酸酶抑制剂提供实验依据和理论基础。
[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.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392;R654.2
本文编号：2211960
[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.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392;R654.2
【参考文献】
相关期刊论文 前1条
1 朱晓星;杨康;吴蔚;;近交系小鼠不同品系颈部心脏移植[J];现代生物医学进展;2008年01期
本文编号：2211960
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