SAHA对T淋巴细胞功能的调控作用及分子机制研究
发布时间：2018-09-01 20:31
【摘要】：研究目的:组蛋白去乙酰化酶抑制剂(HDACI)是一类在染色质水平调控基因表达的化合物,其所调控的基因与细胞周期停滞,细胞分化及细胞凋亡等重要生物学效应密切相关。HDACI对众多实体器官和血液系统肿瘤中具有强大的抗肿瘤活性,因此围绕HDACI的研究主要集中于肿瘤领域。新近研究发现,HDACI具备改善自身免疫性疾病模型症状,调控固有免疫功能以及抑制促炎细胞因子表达等一系列免疫调节活性。T淋巴细胞作为免疫反应的中心环节,在炎症免疫性疾病和移植免疫中发挥重要作用。然而HDACI对T淋巴细胞功能的调控作用和机制及其对移植排斥反应的影响少有报道,其分子机制有待于进一步阐明。我们通过设计体外和体内实验,研究组蛋白去乙酰化酶抑制剂SAHA对T淋巴细胞增殖、活化和分化功能,基因表达调控和移植排斥反应的影响,以探讨HDACI在体外和体内对T淋巴细胞的调控作用及分子机制。研究方法:(1)MACS法体外分选小鼠脾脏来源CD4~+和CD8~+T淋巴细胞,通过ConA或培养板包被的抗CD3/CD28活化,并加入不同浓度SAHA干预,3H-TdR法检测细胞增殖,FCM检测T细胞活化标志分子及细胞凋亡情况,荧光定量PCR检测促炎细胞因子表达,Western-blot分析NF-κB、NFAT等转录因子表达的变化。(2)CD4~+T细胞体外分选及活化同前,流式及荧光定量PCR检测Foxp3表达,分析SAHA对Treg分化的影响;MACS法分选Treg和Teff细胞,分别活化并给予SAHA干预,荧光定量PCR检测FOXP3表达,分析SAHA对Treg体外扩增,Teff转化的作用;CFSE标记的Teff与Treg按照不同比例混合并活化,流式检测增殖情况以分析SAHA对Treg抑制功能的影响,并探讨其分子机制。(3)体外诱导CD4~+T细胞向Th17分化,并加入不同浓度SAHA干预,流式检测IL-17A蛋白表达,荧光定量PCR检测Th17相关基因表达变化,分析SAHA调控Th17分化的分子机制。(4)构建小鼠颈部异位心脏移植模型(BALB/C→C57),观察单独应用SAHA及联合雷帕霉素(RPM)对移植物存活期的影响,取心脏移植物进行病理分析,荧光定量检测移植物内炎性因子表达;移植后第7天流式检测胸腺、脾脏、淋巴结中Treg的比例;过继转移研究SAHA在体内对Teff的转化作用;流式检测SAHA对Treg抑制功能的影响;探讨SAHA对移植排斥反应的调控作用与分子机制。(5)检测SAHA处理组和DMSO处理组小鼠脾脏来源Treg细胞活化前后HDACs表达情况,筛选调控Treg细胞分化的关键HDACs,构建相应siRNA转染Jurkat细胞,荧光定量PCR检测Treg相关基因表达,验证HDACs对Treg细胞分化的调控作用。结果:(1)SAHA对T淋巴细胞增殖、活化和分化的影响: SAHA呈时间及剂量依赖性的抑制CD4~+和CD8~+T淋巴细胞增殖;高浓度SAHA显著抑制CD25和NF-κB表达,而CD69在活化后2h,6h和12h均无显著改变,提示SAHA影响T细胞中后期活化;SAHA阻断IL-2、IFN-γ、IL-12、TNF-α、和IL-10等促炎细胞因子表达;SAHA呈时间及剂量依赖性的促进T细胞凋亡。同时显著抑制体外诱导的Th17分化,提示高浓度SAHA可通过干预T淋巴细胞活化和众多促炎基因表达发挥免疫抑制作用。(2)SAHA在体外对Treg细胞的诱导作用:随着SAHA浓度的增加, CD4~+Foxp3~+T细胞所占的比例显著降低,高浓度SAHA显著下调Foxp3基因表达,而低浓度SAHA(0.1μM)轻度增加了Treg的比例,但没有上调FOXP3表达,流式检测显示低浓度SAHA选择性诱导Teff细胞凋亡,从而间接增加了Treg比例。尽管荧光定量PCR检测显示SAHA在体外不能促进Treg扩增或Teff向Treg转化,但SAHA可通过上调CTLA-4增强Treg的抑制功能。(3)SAHA对Th17分化的影响:流式检测显示SAHA(0.1-1μM)显著抑制IL-17A表达,低浓度SAHA显著抑制IL-17A、IL-17F和STAT3表达,而不影响RORγt,提示SAHA可能通过抑制STAT3通路抑制Th17分化。有趣的是,与对照组相比,SAHA处理后的Th17中FOXP3表达显著上调,显示SAHA可能参与调控Treg和Th17平衡。(4)在小鼠颈部异位心脏移植模型中,载体对照组移植物在7天内即发生排斥反应而停跳,而50mg/kg SAHA显著延长移植物中期存活时间(MST)至16天,而低于治疗剂量的RPM(0.1mg/kg)延长移植物MST至10天,当50mg/kg SAHA与低剂量RPM联合应用时,显著延长移植物MST至26天;组织病理检测显示对照组伴随着心肌结构破坏和间质细胞浸润,SAHA处理组移植物心肌结构仍完整,但间质细胞浸润部分得到改善,而联合用药组既保持了心肌结构完整又阻止了间质细胞浸润。移植物检测发现SAHA处理组Foxp3、CTLA-4、IL-10基因表达显著上调,CD11b、IL-17、INF-γ表达下调,提示SAHA在体内促进Treg分化而抑制Th1和Th17分化。(5)SAHA处理组受者体内胸腺、淋巴结和脾脏中Foxp3~+T细胞比例显著升高,其抑制功能与对照相比显著增强;过继转移实验发现SAHA在体内不能促进外周CD4~+CD25~-T细胞转化为CD4~+Foxp3~+Treg,这些结果表明SAHA在体内增加胸腺来源的天然Treg数量,而非外周转化;在IL-~(2-/-)小鼠受者移植模型中,50mg/kg SAHA无法延长移植物存活时间,由于IL-2对Treg发育必不可少,这些结果提示Treg在SAHA介导的抗移植排斥作用中至关重要。(5)荧光定量PCR检测显示,HDAC1、HDAC2、HDAC3、HDAC7在Treg活化后显著升高,但SAHA处理组与DMSO处理组之间无显著性差异,而HDAC9在活化后显著降低,SAHA处理组进一步下调HDAC9表达,二者之间具有显著性差异,体外通过小siRNA干扰HDAC9可显著上调Foxp3和CTLA4基因表达,提示HDAC9在Treg发育过程中发挥重要作用。结论:本文探讨了SAHA对T淋巴细胞功能和移植排斥反应的调控作用及分子机制。SAHA被证实发挥多种T淋巴细胞调节功能。高浓度SAHA在体外显著促进T细胞凋亡,抑制T淋巴细胞增殖、活化和众多促炎基因表达。而低浓度 SAHA显著增强Treg的抑制功能并可能参与调控Treg和Th17平衡;体内研究显示SAHA通过增加胸腺来源Treg的数量和增强Treg的抑制功能而抑制急性排斥反应的发生。而HDAC9可能对Treg分化和发挥抑制功能具有重要调控作用,这些结果提示SAHA在体内和体外通过不同的机制调控Treg分化,而进一步研究显示HDAC9在Treg分化过程中发挥关键作用。本文证实了SAHA的抗移植排斥作用,并对其机制进行浅析,为HDACI应用于器官移植提供了一定的依据。
[Abstract]:Objective: Histone deacetylase inhibitors (HDACI) are a class of compounds that regulate gene expression at chromatin level. The genes regulated by HDACI are closely related to cell cycle arrest, cell differentiation and apoptosis. Recent studies have found that HDACI has a series of immunomodulatory activities, such as improving the symptoms of autoimmune disease models, regulating innate immune function and inhibiting the expression of pro-inflammatory cytokines. HDACI plays an important role in the epidemic. However, the regulatory effect and mechanism of HDACI on T lymphocyte function and its effect on transplant rejection are seldom reported, and its molecular mechanism needs further elucidation. Methods: (1) MacS method was used to isolate mouse spleen-derived CD4~+ and CD8~+ T lymphocytes in vitro. ConA or plate-coated anti-CD3/CD28 were activated and different concentrations of SAHA were added to interfere with the activation of CD3/CD28. Cell proliferation was detected, T cell activation markers and apoptosis were detected by FCM, pro-inflammatory cytokines were detected by fluorescence quantitative PCR, and transcription factors such as NF-kappa B and NFAT were analyzed by Western blot. Treg and Teff cells were selected by MACS and activated by SAHA respectively. The expression of FOXP3 was detected by fluorescence quantitative PCR, and the effects of SAHA on Treg proliferation and Teff transformation were analyzed. CFSE-labeled Teff and Treg were mixed and activated in different proportions. The proliferation of Treg cells was detected by flow cytometry to analyze the effect of SAHA on Treg inhibition function and explore its molecular mechanism. CD4~+T cells were induced to differentiate into Th17 cells in vitro, and different concentrations of SAHA were added to interfere with it. IL-17A protein expression was detected by flow cytometry, Th17-related gene expression was detected by fluorescence quantitative PCR, and the molecular mechanism of SAHA regulating Th17 differentiation was analyzed. (4) A mouse model of heterotopic heart transplantation was established (BALB/C C57), and SAHA and rapamycin (RP) were used alone. M) Effect of SAHA on graft survival, pathological analysis of heart grafts, quantitative detection of inflammation factor expression in grafts by fluorescence, flow cytometry of Treg in thymus, spleen and lymph nodes on the 7th day after transplantation, adoptive metastasis study of the effect of SAHA on Teff in vivo, flow cytometry of SAHA on Treg inhibition function; (5) To detect the expression of HDACs in spleen-derived Treg cells of SAHA and DMSO treated mice before and after activation, screen the key HDACs that regulate the differentiation of Treg cells, construct the corresponding siRNA to transfect Jurkat cells, detect the expression of Treg-related genes by fluorescence quantitative PCR, and verify the differentiation of Treg cells by HDACs. Results: (1) The effects of SAHA on T lymphocyte proliferation, activation and differentiation: SAHA inhibited the proliferation of CD4~+ and CD8~+ T lymphocytes in a time-and dose-dependent manner; high concentration of SAHA significantly inhibited the expression of CD25 and NF-kappa B, while CD69 did not change significantly at 2, 6 and 12 hours after activation, suggesting that SAHA affected the late activation of T cells; SAHA blocked IL-2. SAHA promoted T cell apoptosis in a time-and dose-dependent manner. SAHA significantly inhibited Th17 differentiation in vitro, suggesting that high concentration of SAHA could exert immunosuppressive effect by interfering with T lymphocyte activation and expression of many pro-inflammatory genes. (2) SAHA could induce Treg cells in vitro. Function: With the increase of SAHA concentration, the percentage of CD4~+Foxp3~+T cells decreased significantly, the expression of Foxp3 gene was significantly down-regulated by high concentration of SAHA, but Treg was slightly increased by low concentration of SAHA (0.1 mu), but FOXP3 expression was not up-regulated. Flow cytometry showed that low concentration of SAHA selectively induced Teff cell apoptosis, thus indirectly increasing Treg ratio. Although fluorescence quantitative PCR assay showed that SAHA could not promote Treg amplification or Teff to Treg transformation in vitro, SAHA could enhance Treg inhibition by up-regulating CTLA-4. (3) The effect of SAHA on Th17 differentiation: Flow cytometry showed that SAHA (0.1-1 muM) significantly inhibited the expression of IL-17A, IL-17F and STAT3, but not ROR. Gamma t, suggesting that SAHA may inhibit Th17 differentiation by inhibiting STAT3 pathway. Interestingly, compared with the control group, FOXP3 expression was significantly up-regulated in Th17 after SAHA treatment, suggesting that SAHA may be involved in regulating the balance of Treg and Th17. (4) In a mouse model of heterotopic heart transplantation in the neck, the graft in the carrier control group stopped beating within 7 days after rejection. 50 mg/kg SAHA significantly prolonged the graft medium-term survival (MST) to 16 days, while RPM (0.1 mg/kg) at lower doses prolonged the graft MST to 10 days. When 50 mg/kg SAHA was combined with low-dose RPM, the graft MST was significantly prolonged to 26 days. Histopathological examination showed that the control group was accompanied by myocardial structural damage and interstitial cell infiltration at SAHA site. The results showed that the expression of Foxp3, CTLA-4, IL-10 and CD11b, IL-17, INF-gamma were significantly up-regulated and down-regulated in SAHA treatment group, suggesting that SAHA could promote Treg differentiation in vivo. (5) The percentage of Foxp3~+ T cells in thymus, lymph nodes and spleen of SAHA treated recipients was significantly increased, and the inhibition function of Foxp3~+ T cells was significantly enhanced compared with the control group. The adoptive metastasis experiment showed that SAHA could not promote the transformation of peripheral CD4~+CD25~-T cells into CD4~+Foxp3~+ Treg in vivo, which indicated that SAHA increased the percentage of thymus in vivo. In the IL-~ (2-/-) mouse recipient transplantation model, 50 mg/kg SAHA could not prolong the survival time of the graft, because IL-2 was essential to the development of Treg, these results suggest that Treg plays an important role in SAHA-mediated anti-graft rejection. (5) Fluorescence quantitative PCR detection showed that HDAC1, HDAC2, HDAC3, HDAC 3, HDAC 7 increased significantly after Treg activation, but there was no significant difference between SAHA treatment group and DMSO treatment group. HDAC9 decreased significantly after activation. The expression of HDAC9 was further down-regulated by SAHA treatment group. There was significant difference between the two groups. HDAC9 gene expression was significantly up-regulated by interfering with HDAC9 by small siRNA in vitro, suggesting that HDAC9 was involved in Treg development. CONCLUSION: SAHA plays an important role in the regulation of T lymphocyte function and graft rejection. SAHA has been proved to play a variety of T lymphocyte regulatory functions. High concentration of SAHA significantly promotes T cell apoptosis, inhibits T lymphocyte proliferation, activates and promotes the expression of many inflammatory genes in vitro.
SAHA significantly enhances Treg inhibition and may be involved in regulating Treg and Th17 homeostasis; in vivo studies have shown that SAHA inhibits acute rejection by increasing the number of Treg derived from thymus and enhancing Treg inhibition. HDAC9 may play an important role in regulating Treg differentiation and inhibiting Treg function. These results suggest that SAHA may be involved in the regulation of Treg differentiation and inhibition in vivo. HDAC9 plays a key role in the differentiation of Treg. This paper confirms the anti-rejection effect of SAHA and analyzes its mechanism, which provides a basis for the application of HDACI in organ transplantation.
【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】：R392.1
本文编号：2218271
[Abstract]:Objective: Histone deacetylase inhibitors (HDACI) are a class of compounds that regulate gene expression at chromatin level. The genes regulated by HDACI are closely related to cell cycle arrest, cell differentiation and apoptosis. Recent studies have found that HDACI has a series of immunomodulatory activities, such as improving the symptoms of autoimmune disease models, regulating innate immune function and inhibiting the expression of pro-inflammatory cytokines. HDACI plays an important role in the epidemic. However, the regulatory effect and mechanism of HDACI on T lymphocyte function and its effect on transplant rejection are seldom reported, and its molecular mechanism needs further elucidation. Methods: (1) MacS method was used to isolate mouse spleen-derived CD4~+ and CD8~+ T lymphocytes in vitro. ConA or plate-coated anti-CD3/CD28 were activated and different concentrations of SAHA were added to interfere with the activation of CD3/CD28. Cell proliferation was detected, T cell activation markers and apoptosis were detected by FCM, pro-inflammatory cytokines were detected by fluorescence quantitative PCR, and transcription factors such as NF-kappa B and NFAT were analyzed by Western blot. Treg and Teff cells were selected by MACS and activated by SAHA respectively. The expression of FOXP3 was detected by fluorescence quantitative PCR, and the effects of SAHA on Treg proliferation and Teff transformation were analyzed. CFSE-labeled Teff and Treg were mixed and activated in different proportions. The proliferation of Treg cells was detected by flow cytometry to analyze the effect of SAHA on Treg inhibition function and explore its molecular mechanism. CD4~+T cells were induced to differentiate into Th17 cells in vitro, and different concentrations of SAHA were added to interfere with it. IL-17A protein expression was detected by flow cytometry, Th17-related gene expression was detected by fluorescence quantitative PCR, and the molecular mechanism of SAHA regulating Th17 differentiation was analyzed. (4) A mouse model of heterotopic heart transplantation was established (BALB/C C57), and SAHA and rapamycin (RP) were used alone. M) Effect of SAHA on graft survival, pathological analysis of heart grafts, quantitative detection of inflammation factor expression in grafts by fluorescence, flow cytometry of Treg in thymus, spleen and lymph nodes on the 7th day after transplantation, adoptive metastasis study of the effect of SAHA on Teff in vivo, flow cytometry of SAHA on Treg inhibition function; (5) To detect the expression of HDACs in spleen-derived Treg cells of SAHA and DMSO treated mice before and after activation, screen the key HDACs that regulate the differentiation of Treg cells, construct the corresponding siRNA to transfect Jurkat cells, detect the expression of Treg-related genes by fluorescence quantitative PCR, and verify the differentiation of Treg cells by HDACs. Results: (1) The effects of SAHA on T lymphocyte proliferation, activation and differentiation: SAHA inhibited the proliferation of CD4~+ and CD8~+ T lymphocytes in a time-and dose-dependent manner; high concentration of SAHA significantly inhibited the expression of CD25 and NF-kappa B, while CD69 did not change significantly at 2, 6 and 12 hours after activation, suggesting that SAHA affected the late activation of T cells; SAHA blocked IL-2. SAHA promoted T cell apoptosis in a time-and dose-dependent manner. SAHA significantly inhibited Th17 differentiation in vitro, suggesting that high concentration of SAHA could exert immunosuppressive effect by interfering with T lymphocyte activation and expression of many pro-inflammatory genes. (2) SAHA could induce Treg cells in vitro. Function: With the increase of SAHA concentration, the percentage of CD4~+Foxp3~+T cells decreased significantly, the expression of Foxp3 gene was significantly down-regulated by high concentration of SAHA, but Treg was slightly increased by low concentration of SAHA (0.1 mu), but FOXP3 expression was not up-regulated. Flow cytometry showed that low concentration of SAHA selectively induced Teff cell apoptosis, thus indirectly increasing Treg ratio. Although fluorescence quantitative PCR assay showed that SAHA could not promote Treg amplification or Teff to Treg transformation in vitro, SAHA could enhance Treg inhibition by up-regulating CTLA-4. (3) The effect of SAHA on Th17 differentiation: Flow cytometry showed that SAHA (0.1-1 muM) significantly inhibited the expression of IL-17A, IL-17F and STAT3, but not ROR. Gamma t, suggesting that SAHA may inhibit Th17 differentiation by inhibiting STAT3 pathway. Interestingly, compared with the control group, FOXP3 expression was significantly up-regulated in Th17 after SAHA treatment, suggesting that SAHA may be involved in regulating the balance of Treg and Th17. (4) In a mouse model of heterotopic heart transplantation in the neck, the graft in the carrier control group stopped beating within 7 days after rejection. 50 mg/kg SAHA significantly prolonged the graft medium-term survival (MST) to 16 days, while RPM (0.1 mg/kg) at lower doses prolonged the graft MST to 10 days. When 50 mg/kg SAHA was combined with low-dose RPM, the graft MST was significantly prolonged to 26 days. Histopathological examination showed that the control group was accompanied by myocardial structural damage and interstitial cell infiltration at SAHA site. The results showed that the expression of Foxp3, CTLA-4, IL-10 and CD11b, IL-17, INF-gamma were significantly up-regulated and down-regulated in SAHA treatment group, suggesting that SAHA could promote Treg differentiation in vivo. (5) The percentage of Foxp3~+ T cells in thymus, lymph nodes and spleen of SAHA treated recipients was significantly increased, and the inhibition function of Foxp3~+ T cells was significantly enhanced compared with the control group. The adoptive metastasis experiment showed that SAHA could not promote the transformation of peripheral CD4~+CD25~-T cells into CD4~+Foxp3~+ Treg in vivo, which indicated that SAHA increased the percentage of thymus in vivo. In the IL-~ (2-/-) mouse recipient transplantation model, 50 mg/kg SAHA could not prolong the survival time of the graft, because IL-2 was essential to the development of Treg, these results suggest that Treg plays an important role in SAHA-mediated anti-graft rejection. (5) Fluorescence quantitative PCR detection showed that HDAC1, HDAC2, HDAC3, HDAC 3, HDAC 7 increased significantly after Treg activation, but there was no significant difference between SAHA treatment group and DMSO treatment group. HDAC9 decreased significantly after activation. The expression of HDAC9 was further down-regulated by SAHA treatment group. There was significant difference between the two groups. HDAC9 gene expression was significantly up-regulated by interfering with HDAC9 by small siRNA in vitro, suggesting that HDAC9 was involved in Treg development. CONCLUSION: SAHA plays an important role in the regulation of T lymphocyte function and graft rejection. SAHA has been proved to play a variety of T lymphocyte regulatory functions. High concentration of SAHA significantly promotes T cell apoptosis, inhibits T lymphocyte proliferation, activates and promotes the expression of many inflammatory genes in vitro.
SAHA significantly enhances Treg inhibition and may be involved in regulating Treg and Th17 homeostasis; in vivo studies have shown that SAHA inhibits acute rejection by increasing the number of Treg derived from thymus and enhancing Treg inhibition. HDAC9 may play an important role in regulating Treg differentiation and inhibiting Treg function. These results suggest that SAHA may be involved in the regulation of Treg differentiation and inhibition in vivo. HDAC9 plays a key role in the differentiation of Treg. This paper confirms the anti-rejection effect of SAHA and analyzes its mechanism, which provides a basis for the application of HDACI in organ transplantation.
【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】：R392.1
【共引文献】
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