發(fā)布時(shí)間：2018-01-16 06:10

  本文關(guān)鍵詞：Tim-4在調(diào)控Kupffer細(xì)胞免疫功能及誘導(dǎo)肝移植術(shù)后免疫耐受中的機(jī)制研究　出處：《重慶醫(yī)科大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： T細(xì)胞免疫球蛋白黏蛋白-4 肝臟移植 枯否氏細(xì)胞 急性排斥反應(yīng) 免疫耐受

【摘要】：背景肝移植是目前治療各種終末期肝病的最佳治療手段。而移植術(shù)后急、慢性排斥反應(yīng)、缺血再灌注損傷,與肝臟供體缺乏一起,成為了肝移植領(lǐng)域面臨的主要挑戰(zhàn)。誘導(dǎo)肝移植術(shù)后免疫耐受是避免移植肝臟失功及長(zhǎng)期服用免疫抑制劑的最佳途徑,也是近年移植領(lǐng)域研究的重點(diǎn)和熱點(diǎn)。Kupffer細(xì)胞(KCs)作為肝臟的最大巨噬細(xì)胞群,在肝移植免疫中發(fā)揮著極其重要的作用,其機(jī)制與抗原提呈、不同表型下的免疫功能及調(diào)控T淋巴細(xì)胞免疫功能等密切相關(guān)。新近研究發(fā)現(xiàn),T細(xì)胞免疫球蛋白粘蛋白4(T cell immunoglobulin domain and mucin domain,Tim-4)參與了Th2細(xì)胞分化、凋亡細(xì)胞吞噬清除、巨噬細(xì)胞功能調(diào)節(jié)等諸多免疫調(diào)控的重要環(huán)節(jié)。但目前為止有關(guān)Tim-4分子的研究主要集中在變態(tài)反應(yīng)性疾病及自身免疫性疾病方面,尚未涉及肝移植免疫。因此,探索Tim-4對(duì)KCs免疫功能及在肝移植術(shù)后免疫調(diào)控中的作用及機(jī)制,可能為誘導(dǎo)肝移植術(shù)后免疫耐受提供新的思路和理論依據(jù)。第一部分Tim-4表達(dá)變化對(duì)Kupffer細(xì)胞免疫功能調(diào)控的作用研究目的通過(guò)抑制或上調(diào)KCs中Tim-4表達(dá),觀察Tim-4過(guò)表達(dá)及沉默狀態(tài)下對(duì)KCs分泌Th1/Th2類(lèi)細(xì)胞因子及抗原呈遞作用的影響及相關(guān)機(jī)制。方法(1)非連續(xù)梯度離心法分離、培養(yǎng)BALB/c小鼠肝臟KCs,免疫熒光染色及臺(tái)盼藍(lán)染色判斷細(xì)胞純度及活率,吞墨實(shí)驗(yàn)判斷其吞噬功能;(2)流式細(xì)胞儀及免疫組化檢測(cè)KCs特異性表面分子CD163和CD68的表達(dá)來(lái)鑒定KCs;(3)鑒定后的KCs隨機(jī)分為四組:Tim-4過(guò)表達(dá)組(Ad V-Tim4 group);過(guò)表達(dá)對(duì)照組(Ctrl-Ad V group);Tim-4抑制組(Tim-4 sh RNA group);抑制對(duì)照組(Ctrl-sh RNA group);分別轉(zhuǎn)染Tim-4過(guò)表達(dá)、抑制及相應(yīng)錯(cuò)義序列慢病毒獲得穩(wěn)定封閉的細(xì)胞群。以LPS(終濃度100ng/m L)活化KCs后作KCs免疫功能相關(guān)檢測(cè);(4)免疫熒光及共聚焦顯微鏡檢測(cè)評(píng)估Tim-4相關(guān)慢病毒轉(zhuǎn)染效率;(5)ELISA檢測(cè)細(xì)胞培養(yǎng)液中TNF-α、IL-1β、IFN-γ、IL-10、TGF-β表達(dá)水平;(6)Western Blot及RT-PCR檢測(cè)各組KCs中Tim-4、TNF-α、IL-1β、IFN-γ、IL-10、TGF-β蛋白和基因表達(dá)水平;(7)流式細(xì)胞術(shù)(FCM)檢測(cè)各組KCs的表型分子表達(dá)情況;(8)分離BALB/c小鼠脾細(xì)胞懸液,制備純化T細(xì)胞,FCM檢測(cè)純度,與上述各組KCs共培養(yǎng),MTT法檢測(cè)T細(xì)胞增殖情況;Annexin V/PI法觀察T淋巴細(xì)胞凋亡情況;(10)ELISA檢測(cè)T細(xì)胞培養(yǎng)液中TNF-α、IL-1β、IFN-γ、IL-10、TGF-β等細(xì)胞因子含量;(11)Western-blot測(cè)定KCs中LPS/TLR4/NF-κB及MAPK通路關(guān)鍵蛋白p65、p38、ERK1/2、JNK、IRF3及其磷酸化蛋白表達(dá)水平。結(jié)果(1)KCs細(xì)胞表面特異性分子表達(dá)呈陽(yáng)性,臺(tái)盼藍(lán)染色提示KCs活力90%,吞墨實(shí)驗(yàn)證實(shí)其具有良好的吞噬能力;(2)轉(zhuǎn)染Tim-4慢病毒后,6h開(kāi)始KCs即出現(xiàn)熒光表達(dá),48h前后達(dá)到高峰;Western Blot檢測(cè)結(jié)果顯示:Ad V-Tim4組中Tim-4蛋白水平顯著高于對(duì)照組(1.90±0.41 vs.1.13±0.32,P0.05);而Tim-4 sh RNA組中Tim-4的蛋白表達(dá)水平與其對(duì)照組相比較顯著降低(0.52±0.11 vs.1.3±0.31,P0.05);(3)FCM檢測(cè)活化的KCs表面共刺激發(fā)現(xiàn):Ad V-Tim4中MHC-II、CD80、CD86、CD40的表達(dá)明顯低于對(duì)照組,而M2型分子CD204和CD206的表達(dá)則高于對(duì)照組;而在Tim-4 sh RNA組中,以上分子的表達(dá)則呈相反趨勢(shì);(4)ELISA檢測(cè)各組KCs培養(yǎng)液中細(xì)胞因子發(fā)現(xiàn):在Ad V-Tim4組,TNF-α、IL-1β、IFN-γ的表達(dá)與其對(duì)照組比較明顯降低(P0.05),而IL-10、TGF-β水平較對(duì)照組表達(dá)增高;在Tim-4抑制組,TNF-α、IL-1β、IFN-γ的表達(dá)較其對(duì)照組增高,而IL-10、TGF-β的表達(dá)量則呈相反趨勢(shì)(P0.05);同時(shí),Western Blot證實(shí)KCs中上述細(xì)胞因子的蛋白水平變化與ELISA結(jié)果一致。(5)Western Blot檢測(cè)發(fā)現(xiàn):Ad V-Tim4組中KCs內(nèi)Ikkα,IκBα,NF-κB p65,ERKl/2、p38和IRF3的蛋白表達(dá)及其磷酸化水平均受到抑制,JNK蛋白表達(dá)無(wú)顯著變化;而在Tim-4 sh RNA中,上述蛋白的磷酸化表達(dá)有所增強(qiáng);(5)T細(xì)胞與各組KCs共培養(yǎng)72h后,MTT結(jié)果表明:Ad V-Tim4組T淋巴細(xì)胞的增殖較對(duì)照組受到抑制(0.43±0.04 vs.0.72±0.05,P0.05);而Tim-4 sh RNA組中T淋巴細(xì)胞增殖有所增強(qiáng)(0.92±0.07 vs.0.69±0.06,P0.05);(6)FCM檢測(cè)發(fā)現(xiàn)Ad V-Tim4組中T淋巴細(xì)胞凋亡明顯增加(38.42%±7.36%),而抑制Tim-4后T淋巴細(xì)胞凋亡率顯著下降(13.98%±4.59%);(7)ELISA檢測(cè)發(fā)現(xiàn):Ad V-Tim4組與T細(xì)胞培養(yǎng)液中促炎細(xì)胞因子(TNF-α、IL-1β、IFN-γ)分泌減少,抗炎細(xì)胞因子(IL-10)分泌增加(P0.05),而Tim-4抑制組呈相反趨勢(shì)。結(jié)論(1)過(guò)表達(dá)Tim-4可調(diào)控活化的KCs分泌譜改變,其機(jī)制可能與Tim-4抑制了LPS/TLR4/NF-k B、LPS/MAPK及IRF3通路活性有關(guān);(2)過(guò)表達(dá)KCs中Tim-4可抑制KCs表面共刺激分子(MHC-II、CD80、CD86、CD40等)的表達(dá),同時(shí)M2型KCs表型分子表達(dá)增加;(3)過(guò)表達(dá)Tim-4能有效抑制KCs的抗原提呈功能,抑制T淋巴細(xì)胞的增殖及促進(jìn)活化T細(xì)胞凋亡。第二部分KCs中Tim-4表達(dá)變化在小鼠肝移植免疫耐受誘導(dǎo)的作用研究目的經(jīng)門(mén)靜脈灌注供肝轉(zhuǎn)染過(guò)表達(dá)Tim-4或Tim-4-sh RNA慢病毒,觀察增強(qiáng)或抑制KCs中Tim-4表達(dá)對(duì)肝移植后Ac R程度的影響,并探討其相關(guān)機(jī)制。方法(1)采用改良“雙袖套”法建立BALB/c→C3H小鼠肝移植急性排斥反應(yīng)模型;(2)受體隨機(jī)分為三組(n=30只/組):Tim-4過(guò)表達(dá)組(Ad V-Tim4 group);Tim-4抑制組(Tim-4 sh RNA group);錯(cuò)義序列對(duì)照組(control group)。分別于供肝血管吻合完成后經(jīng)門(mén)靜脈高壓注射攜帶Tim-4的慢病毒感染供肝;(3)每組取10只動(dòng)物觀察各組術(shù)后生存時(shí)間及生存率,其余受體于術(shù)后7d獲取組織及血液標(biāo)本;(4)ELISA法檢測(cè)血液中TNF-α、IL-1β、IFN-γ、IL-10及TGF-β含量,Real-time PCR檢測(cè)肝組織中上述細(xì)胞因子的m RNA表達(dá)水平,全自動(dòng)生化儀檢測(cè)轉(zhuǎn)氨酶及膽紅素水平;(5)Western-blot測(cè)定肝臟KCs中Tim-4、p65、p38、ERK1/2、JNK、IRF3及其磷酸化蛋白表達(dá)水平;(6)獲取光、電鏡標(biāo)本觀察病理形態(tài)變化;免疫組織化學(xué)染色及激光共聚焦觀察各組移植物中CD4+T細(xì)胞和KCs的浸潤(rùn)情況;TUNEL檢測(cè)移植區(qū)T細(xì)胞凋亡情況;透射電鏡和激光共聚焦顯微鏡判斷KCs吞噬凋亡細(xì)胞情況。結(jié)果(1)術(shù)后第7天,Ad V-Tim4組受體生存率以及肝功能較其余兩組得到明顯的改善(P0.05);(2)HE染色表明Ad V-Tim4組RAI評(píng)分屬輕度排斥反應(yīng),其余兩組則呈重度排斥反應(yīng);(3)PCR結(jié)果發(fā)現(xiàn),細(xì)胞因子IL-1β、TNF-α、IFN-γ的m RNA水平在Ad V-Tim4組低表達(dá),而IL-10呈高表達(dá)(P0.05);外周血ELISA檢測(cè)結(jié)果與PCR結(jié)果一致;(4)Western-blot檢測(cè)到Ad V-Tim4組肝臟KCs中Tim-4表達(dá)較Control組和Tim4-sh RNA組增高(P0.05),但p65、p38、ERK1/2、JNK和IRF3的磷酸化蛋白表達(dá)水平更低(P0.05);(5)TUNEL顯示Ad V-Tim4組匯管區(qū)淋巴細(xì)胞凋亡率遠(yuǎn)高于Control組和Tim4-sh RNA組;(6)免疫組化、激光共聚焦結(jié)果提示:與對(duì)照組相比,CD4+T細(xì)胞及活化KCs在Ad V-Tim4組浸潤(rùn)減少,而在Tim4-sh RNA組顯著增加;(7)激光共聚焦顯示KCs在Ad V-Tim4組吞噬凋亡細(xì)胞顯著增多,而Tim4-sh RNA組KCs吞噬明顯減少。結(jié)論(1)在受體內(nèi)上調(diào)KCs中Tim-4表達(dá)能顯著減輕肝移植后Ac R對(duì)肝臟組織的損傷程度并誘導(dǎo)免疫耐受形成;(2)其機(jī)制可能與Tim-4抑制KCs內(nèi)LPS/TLR4/NF-k B、LPS/MAPK及IRF3等信號(hào)通路活性、減少促炎細(xì)胞因子分泌、減少移植肝臟內(nèi)CD4+T細(xì)胞局部浸潤(rùn)并促進(jìn)其凋亡、同時(shí)促進(jìn)KCs對(duì)凋亡細(xì)胞的吞噬清除等因素有關(guān)。
[Abstract]:Liver transplantation is the treatment of end-stage liver disease the best treatment. But after transplantation of acute, chronic rejection, ischemia reperfusion injury, and the lack of donor liver, has become a major challenge in the field of liver transplantation. To induce immune tolerance after liver transplantation is to avoid liver graft dysfunction and the best way of long-term immunosuppressant drugs, is also the focus of research in the field of transplantation and hot.Kupffer cells (KCs) as the largest group of liver macrophages, play an extremely important role in immune liver transplantation, and the mechanism of antigen presentation, different phenotypes under the immune function and regulation of immune function of T lymphocytes and other closely related. A recent study found, T cell immunoglobulin mucin 4 (T cell immunoglobulin domain and mucin domain, Tim-4) in the differentiation of Th2 cells and phagocytic clearance of apoptotic cells, macrophage function adjustment An important part of festival and many other immune regulation. But the research so far about Tim-4 molecules mainly focus on allergic diseases and autoimmune diseases, has not been involved in immune liver transplantation. Therefore, exploring the Tim-4 KCs on immune function and immune regulation in the patients after liver transplantation and its action mechanism, may provide a new methods and theoretical basis for induction of immune tolerance after liver transplantation. The purpose of the first part of the study of regulation of Tim-4 expression changes of Kupffer cell immune function by inhibiting or Tim-4 upregulation of KCs expression, Tim-4 overexpression was observed and the effect of silencing condition on KCs secretion of Th1/Th2 cytokines and antigen presenting function and related mechanism. Methods (1) separation discontinuous gradient centrifugation and cultured BALB/c mouse liver KCs, immunofluorescence staining and trypan blue staining to determine the cell purity and viability, swallow ink assays to detect phagocytosis ability ;(2)嫻佸紡緇嗚優(yōu)浠�鍙�(qiáng)鍏嶇柅緇勫寲媯，

本文編號(hào)：1431861