【摘要】：研究目的:組蛋白去乙�；敢种苿�(HDACI)是一類在染色質(zhì)水平調(diào)控基因表達的化合物,其所調(diào)控的基因與細胞周期停滯,細胞分化及細胞凋亡等重要生物學效應密切相關(guān)。HDACI對眾多實體器官和血液系統(tǒng)腫瘤中具有強大的抗腫瘤活性,因此圍繞HDACI的研究主要集中于腫瘤領(lǐng)域。新近研究發(fā)現(xiàn),HDACI具備改善自身免疫性疾病模型癥狀,調(diào)控固有免疫功能以及抑制促炎細胞因子表達等一系列免疫調(diào)節(jié)活性。T淋巴細胞作為免疫反應的中心環(huán)節(jié),在炎癥免疫性疾病和移植免疫中發(fā)揮重要作用。然而HDACI對T淋巴細胞功能的調(diào)控作用和機制及其對移植排斥反應的影響少有報道,其分子機制有待于進一步闡明。我們通過設(shè)計體外和體內(nèi)實驗,研究組蛋白去乙�；敢种苿㏒AHA對T淋巴細胞增殖、活化和分化功能,基因表達調(diào)控和移植排斥反應的影響,以探討HDACI在體外和體內(nèi)對T淋巴細胞的調(diào)控作用及分子機制。研究方法:(1)MACS法體外分選小鼠脾臟來源CD4~+和CD8~+T淋巴細胞,通過ConA或培養(yǎng)板包被的抗CD3/CD28活化,并加入不同濃度SAHA干預,3H-TdR法檢測細胞增殖,FCM檢測T細胞活化標志分子及細胞凋亡情況,熒光定量PCR檢測促炎細胞因子表達,Western-blot分析NF-κB、NFAT等轉(zhuǎn)錄因子表達的變化。(2)CD4~+T細胞體外分選及活化同前,流式及熒光定量PCR檢測Foxp3表達,分析SAHA對Treg分化的影響;MACS法分選Treg和Teff細胞,分別活化并給予SAHA干預,熒光定量PCR檢測FOXP3表達,分析SAHA對Treg體外擴增,Teff轉(zhuǎn)化的作用;CFSE標記的Teff與Treg按照不同比例混合并活化,流式檢測增殖情況以分析SAHA對Treg抑制功能的影響,并探討其分子機制。(3)體外誘導CD4~+T細胞向Th17分化,并加入不同濃度SAHA干預,流式檢測IL-17A蛋白表達,熒光定量PCR檢測Th17相關(guān)基因表達變化,分析SAHA調(diào)控Th17分化的分子機制。(4)構(gòu)建小鼠頸部異位心臟移植模型(BALB/C→C57),觀察單獨應用SAHA及聯(lián)合雷帕霉素(RPM)對移植物存活期的影響,取心臟移植物進行病理分析,熒光定量檢測移植物內(nèi)炎性因子表達;移植后第7天流式檢測胸腺、脾臟、淋巴結(jié)中Treg的比例;過繼轉(zhuǎn)移研究SAHA在體內(nèi)對Teff的轉(zhuǎn)化作用;流式檢測SAHA對Treg抑制功能的影響;探討SAHA對移植排斥反應的調(diào)控作用與分子機制。(5)檢測SAHA處理組和DMSO處理組小鼠脾臟來源Treg細胞活化前后HDACs表達情況,篩選調(diào)控Treg細胞分化的關(guān)鍵HDACs,構(gòu)建相應siRNA轉(zhuǎn)染Jurkat細胞,熒光定量PCR檢測Treg相關(guān)基因表達,驗證HDACs對Treg細胞分化的調(diào)控作用。結(jié)果:(1)SAHA對T淋巴細胞增殖、活化和分化的影響: SAHA呈時間及劑量依賴性的抑制CD4~+和CD8~+T淋巴細胞增殖;高濃度SAHA顯著抑制CD25和NF-κB表達,而CD69在活化后2h,6h和12h均無顯著改變,提示SAHA影響T細胞中后期活化;SAHA阻斷IL-2、IFN-γ、IL-12、TNF-α、和IL-10等促炎細胞因子表達;SAHA呈時間及劑量依賴性的促進T細胞凋亡。同時顯著抑制體外誘導的Th17分化,提示高濃度SAHA可通過干預T淋巴細胞活化和眾多促炎基因表達發(fā)揮免疫抑制作用。(2)SAHA在體外對Treg細胞的誘導作用:隨著SAHA濃度的增加, CD4~+Foxp3~+T細胞所占的比例顯著降低,高濃度SAHA顯著下調(diào)Foxp3基因表達,而低濃度SAHA(0.1μM)輕度增加了Treg的比例,但沒有上調(diào)FOXP3表達,流式檢測顯示低濃度SAHA選擇性誘導Teff細胞凋亡,從而間接增加了Treg比例。盡管熒光定量PCR檢測顯示SAHA在體外不能促進Treg擴增或Teff向Treg轉(zhuǎn)化,但SAHA可通過上調(diào)CTLA-4增強Treg的抑制功能。(3)SAHA對Th17分化的影響:流式檢測顯示SAHA(0.1-1μM)顯著抑制IL-17A表達,低濃度SAHA顯著抑制IL-17A、IL-17F和STAT3表達,而不影響RORγt,提示SAHA可能通過抑制STAT3通路抑制Th17分化。有趣的是,與對照組相比,SAHA處理后的Th17中FOXP3表達顯著上調(diào),顯示SAHA可能參與調(diào)控Treg和Th17平衡。(4)在小鼠頸部異位心臟移植模型中,載體對照組移植物在7天內(nèi)即發(fā)生排斥反應而停跳,而50mg/kg SAHA顯著延長移植物中期存活時間(MST)至16天,而低于治療劑量的RPM(0.1mg/kg)延長移植物MST至10天,當50mg/kg SAHA與低劑量RPM聯(lián)合應用時,顯著延長移植物MST至26天;組織病理檢測顯示對照組伴隨著心肌結(jié)構(gòu)破壞和間質(zhì)細胞浸潤,SAHA處理組移植物心肌結(jié)構(gòu)仍完整,但間質(zhì)細胞浸潤部分得到改善,而聯(lián)合用藥組既保持了心肌結(jié)構(gòu)完整又阻止了間質(zhì)細胞浸潤。移植物檢測發(fā)現(xiàn)SAHA處理組Foxp3、CTLA-4、IL-10基因表達顯著上調(diào),CD11b、IL-17、INF-γ表達下調(diào),提示SAHA在體內(nèi)促進Treg分化而抑制Th1和Th17分化。(5)SAHA處理組受者體內(nèi)胸腺、淋巴結(jié)和脾臟中Foxp3~+T細胞比例顯著升高,其抑制功能與對照相比顯著增強;過繼轉(zhuǎn)移實驗發(fā)現(xiàn)SAHA在體內(nèi)不能促進外周CD4~+CD25~-T細胞轉(zhuǎn)化為CD4~+Foxp3~+Treg,這些結(jié)果表明SAHA在體內(nèi)增加胸腺來源的天然Treg數(shù)量,而非外周轉(zhuǎn)化;在IL-~(2-/-)小鼠受者移植模型中,50mg/kg SAHA無法延長移植物存活時間,由于IL-2對Treg發(fā)育必不可少,這些結(jié)果提示Treg在SAHA介導的抗移植排斥作用中至關(guān)重要。(5)熒光定量PCR檢測顯示,HDAC1、HDAC2、HDAC3、HDAC7在Treg活化后顯著升高,但SAHA處理組與DMSO處理組之間無顯著性差異,而HDAC9在活化后顯著降低,SAHA處理組進一步下調(diào)HDAC9表達,二者之間具有顯著性差異,體外通過小siRNA干擾HDAC9可顯著上調(diào)Foxp3和CTLA4基因表達,提示HDAC9在Treg發(fā)育過程中發(fā)揮重要作用。結(jié)論:本文探討了SAHA對T淋巴細胞功能和移植排斥反應的調(diào)控作用及分子機制。SAHA被證實發(fā)揮多種T淋巴細胞調(diào)節(jié)功能。高濃度SAHA在體外顯著促進T細胞凋亡,抑制T淋巴細胞增殖、活化和眾多促炎基因表達。而低濃度 SAHA顯著增強Treg的抑制功能并可能參與調(diào)控Treg和Th17平衡;體內(nèi)研究顯示SAHA通過增加胸腺來源Treg的數(shù)量和增強Treg的抑制功能而抑制急性排斥反應的發(fā)生。而HDAC9可能對Treg分化和發(fā)揮抑制功能具有重要調(diào)控作用,這些結(jié)果提示SAHA在體內(nèi)和體外通過不同的機制調(diào)控Treg分化,而進一步研究顯示HDAC9在Treg分化過程中發(fā)揮關(guān)鍵作用。本文證實了SAHA的抗移植排斥作用,并對其機制進行淺析,為HDACI應用于器官移植提供了一定的依據(jù)。
[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.
【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】：R392.1

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