本文關(guān)鍵詞：骨髓間充質(zhì)干細(xì)胞對(duì)慢加急性肝衰竭小鼠調(diào)節(jié)性B細(xì)胞的影響　出處：《中山大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 慢加急性肝衰竭 調(diào)節(jié)性B細(xì)胞 骨髓間充質(zhì)干細(xì)胞 免疫調(diào)節(jié) IL-10

【摘要】：【背景】肝功能衰竭(Liver failure)是多種因素導(dǎo)致的肝臟解毒、合成、排泄、生物轉(zhuǎn)化等功能障礙,病死率在60%以上。亞太地區(qū)以慢加急性肝衰竭(Acute on chronic liver failure,ACLF)多見(jiàn)。肝衰竭目前最有效的治療方法為肝移植,但肝移植受限于供肝缺乏、排斥反應(yīng)、巨大經(jīng)濟(jì)負(fù)擔(dān)等多方面因素,難以廣泛應(yīng)用,而間充質(zhì)干細(xì)胞(Marrow mesenchymal stem cell,MSC)移植治療作為一種新型治療方法具有較好應(yīng)用前景。前期大量研究證實(shí),MSC同時(shí)具有多向分化、免疫調(diào)節(jié)等功能,但MSC在體內(nèi)的免疫調(diào)節(jié)作用機(jī)制研究較少,尤其是MSC移植術(shù)治療ACLF的免疫調(diào)節(jié)作用機(jī)制少有報(bào)道。調(diào)節(jié)性B細(xì)胞(Regulatory B cell,Breg)作為免疫調(diào)節(jié)網(wǎng)絡(luò)的重要組成部分,是一群具有調(diào)節(jié)免疫應(yīng)答、抑制炎癥反應(yīng)等功能的細(xì)胞亞群,其在MSC治療ACLF時(shí)免疫調(diào)節(jié)作用相關(guān)研究較少。因此,本研究旨在對(duì)Breg細(xì)胞在ACLF免疫失衡中的作用及Breg細(xì)胞在MSC治療ACLF時(shí)如何參與免疫調(diào)節(jié)進(jìn)行初步探討。【目的】1.研究Breg細(xì)胞在慢加急性肝衰竭的小鼠免疫失衡的作用。2.探討MSC移植治療ACLF小鼠過(guò)程中,Breg細(xì)胞是否參與MSC免疫調(diào)節(jié)功能�！静牧吓c方法】1.連續(xù)8周腹腔注射四氯化碳的方法誘導(dǎo)慢性肝纖維化模型,此后給予單次亞致死量四氯化碳造成急性肝臟損傷,構(gòu)建ACLF的小鼠疾病模型,觀察對(duì)比疾病組與對(duì)照組的Breg細(xì)胞的表達(dá)和功能。2.分離、培養(yǎng)MSCs,經(jīng)尾靜脈注射MSCs細(xì)胞懸液治療ACLF小鼠,觀察24h累計(jì)生存率。3.分別采集對(duì)照組、ACLF組、MSC治療組小鼠的肝臟、脾臟、外周血,分離提取其中的淋巴細(xì)胞,加入PMA+Ionomycin+Monensin+LPS(PIM+L)刺激5h,流式細(xì)胞術(shù)檢測(cè)CD19+IL-10+B細(xì)胞比例,并采用ELISA檢測(cè)淋巴細(xì)胞培養(yǎng)液中IL-10表達(dá)水平。【結(jié)果】1.ACLF組小鼠的24h累積生存率較對(duì)照組小鼠降低(20%VS 100%,P0.001)。2.ACLF小鼠肝臟、脾臟、外周血的CD19+IL-10+B細(xì)胞表達(dá)頻率高于對(duì)照組,分別為(2.97±0.33)%Vs(0.75±0.11)%,(4.13±0.67)%Vs(0.79±0.11)%,(5.87±1.00)%Vs(0.68±0.09)%,P值均小于0.001。3.ACLF小鼠肝臟、脾臟、外周血IL-10的分泌水平高于對(duì)照組,分別為(16.76±1.73)Vs(6.29±0.70)pg/ml,(21.21±1.62)Vs(8.23±1.52)pg/ml,(31.32±2.95)4.Vs(7.49±1.22)pg/ml,P值均小于0.001。5.MSC治療組小鼠24h累計(jì)生存率高于ACLF組小鼠(60%VS 10%,P0.05)。6.MSC治療組小鼠肝臟、脾臟、外周血的CD19+IL-10+B細(xì)胞表達(dá)頻率低于ACLF組,分別為(2.27±0.17)%Vs(3.67±0.21)%,P0.001,(2.57±0.25)%Vs(5.14±0.59)%,P0.05,(3.00±0.24)%Vs(5.93±0.99)%,P0.05。7.MSC治療小鼠肝臟、脾臟、外周血IL-10的分泌水平低于ACLF組小鼠,分別為(14.08±2.04)Vs(19.97±1.06)pg/ml,(16.30±2.22)Vs(23.41±1.41)pg/ml,(18.05±2.39)Vs(33.21±3.31)pg/ml,差異有統(tǒng)計(jì)學(xué)意義(t=2.458,P0.05;t=2.77,P0.05;t=3.53,P0.01)。【結(jié)論】1.慢加急性肝衰竭小鼠的24h累計(jì)存活率降低,其肝臟、脾臟、外周血的Breg細(xì)胞表達(dá)頻率及IL-10的表達(dá)高于對(duì)照組,Breg在慢加急性肝衰竭小鼠疾病進(jìn)展中起重要作用。2.MSC移植治療可提高ACLF小鼠生存率,可能與MSC下調(diào)Breg數(shù)量以及IL-10分泌有關(guān)。
[Abstract]:[background] liver failure (Liver failure) is caused by a variety of factors of liver detoxification, synthesis, excretion, biotransformation and dysfunction, the death rate is over 60%. The Asia Pacific region with acute on chronic liver failure (Acute on chronic liver failure, ACLF). The treatment of liver failure. Methods the most effective for the liver transplantation, but liver transplantation is limited by lack of donor liver rejection, many factors such as huge economic burden, is difficult to be widely applied, and mesenchymal stem cells (Marrow mesenchymal stem cell, MSC) transplantation as a new therapeutic method has good application prospect. Previous studies confirmed that MSC and multilineage differentiation the function, such as immune regulation, but the regulation mechanism of MSC less in vivo immune, especially immune MSC transplantation in the treatment of ACLF regulatory mechanisms are rarely reported. Regulatory B cells (Regulatory B cell, Breg) as An important part of immune regulation network, is a group of regulating immune response, inhibiting inflammation and other functions of the cell subsets, less research on the role of immune regulation in the treatment of MSC ACLF. Therefore, this study aims to the role of Breg cells in the immune imbalance of ACLF and Breg in MSC cells in the treatment of ACLF in immune regulation was discussed. [Objective].2. 1. mice immune imbalance of Breg cells in acute on chronic liver failure and explore the transplantation of MSC ACLF mice, Breg cells involved in the immune function of MSC. Chronic liver fibrosis model induced by methods [materials and methods] 1. consecutive 8 weeks after intraperitoneal injection of CCl4. Give a single sub lethal dose of carbon tetrachloride induced acute liver injury in mice model of ACLF disease, to observe the expression and function between disease group and control group Breg cells can Separation of.2., culture MSCs, intravenous injection of MSCs cell suspension in the treatment of ACLF mice, observe the 24h cumulative survival rate of.3. were collected as control group, ACLF group, MSC treatment group, the liver, spleen, peripheral blood, extracted from the lymphocytes, adding PMA+Ionomycin+Monensin+ (PIM+L) LPS stimulated 5h, CD19+IL-10+B cell ratio detection flow cytometry, and ELISA was used to detect the expression level of IL-10 lymphocyte culture fluid. [result] 1.ACLF mice 24h cumulative survival rate than the control group mice decreased (20%VS 100%, P0.001).2.ACLF mice liver, spleen, peripheral blood CD19+IL-10+B cells expression frequency was higher than the control group, respectively (2.97 + 0.33)%Vs (0.75 + 0.11)% and (4.13 + 0.67)%Vs (0.79 + 0.11)% and (5.87 + 1)%Vs (0.68 + 0.09)%, P values were less than the liver and spleen of 0.001.3.ACLF mice, the secretion level of IL-10 in peripheral blood was higher than the control group, respectively (16.76 + 1.73) V s(6.29鹵0.70)pg/ml,(21.21鹵1.62)Vs(8.23鹵1.52)pg/ml,(31.32鹵2.95)4.Vs(7.49鹵1.22)pg/ml,P鍊煎潎灝忎簬0.001.5.MSC娌葷枟緇勫皬榧，

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