骨髓间充质干细胞对慢加急性肝衰竭小鼠调节性B细胞的影响

发布时间：2018-01-15 07:24

  本文关键词：骨髓间充质干细胞对慢加急性肝衰竭小鼠调节性B细胞的影响　出处：《中山大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： 慢加急性肝衰竭 调节性B细胞 骨髓间充质干细胞 免疫调节 IL-10

【摘要】：【背景】肝功能衰竭(Liver failure)是多种因素导致的肝脏解毒、合成、排泄、生物转化等功能障碍,病死率在60%以上。亚太地区以慢加急性肝衰竭(Acute on chronic liver failure,ACLF)多见。肝衰竭目前最有效的治疗方法为肝移植,但肝移植受限于供肝缺乏、排斥反应、巨大经济负担等多方面因素,难以广泛应用,而间充质干细胞(Marrow mesenchymal stem cell,MSC)移植治疗作为一种新型治疗方法具有较好应用前景。前期大量研究证实,MSC同时具有多向分化、免疫调节等功能,但MSC在体内的免疫调节作用机制研究较少,尤其是MSC移植术治疗ACLF的免疫调节作用机制少有报道。调节性B细胞(Regulatory B cell,Breg)作为免疫调节网络的重要组成部分,是一群具有调节免疫应答、抑制炎症反应等功能的细胞亚群,其在MSC治疗ACLF时免疫调节作用相关研究较少。因此,本研究旨在对Breg细胞在ACLF免疫失衡中的作用及Breg细胞在MSC治疗ACLF时如何参与免疫调节进行初步探讨。【目的】1.研究Breg细胞在慢加急性肝衰竭的小鼠免疫失衡的作用。2.探讨MSC移植治疗ACLF小鼠过程中,Breg细胞是否参与MSC免疫调节功能。【材料与方法】1.连续8周腹腔注射四氯化碳的方法诱导慢性肝纤维化模型,此后给予单次亚致死量四氯化碳造成急性肝脏损伤,构建ACLF的小鼠疾病模型,观察对比疾病组与对照组的Breg细胞的表达和功能。2.分离、培养MSCs,经尾静脉注射MSCs细胞悬液治疗ACLF小鼠,观察24h累计生存率。3.分别采集对照组、ACLF组、MSC治疗组小鼠的肝脏、脾脏、外周血,分离提取其中的淋巴细胞,加入PMA+Ionomycin+Monensin+LPS(PIM+L)刺激5h,流式细胞术检测CD19+IL-10+B细胞比例,并采用ELISA检测淋巴细胞培养液中IL-10表达水平。【结果】1.ACLF组小鼠的24h累积生存率较对照组小鼠降低(20%VS 100%,P0.001)。2.ACLF小鼠肝脏、脾脏、外周血的CD19+IL-10+B细胞表达频率高于对照组,分别为(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小鼠肝脏、脾脏、外周血IL-10的分泌水平高于对照组,分别为(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治疗组小鼠24h累计生存率高于ACLF组小鼠(60%VS 10%,P0.05)。6.MSC治疗组小鼠肝脏、脾脏、外周血的CD19+IL-10+B细胞表达频率低于ACLF组,分别为(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治疗小鼠肝脏、脾脏、外周血IL-10的分泌水平低于ACLF组小鼠,分别为(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,差异有统计学意义(t=2.458,P0.05;t=2.77,P0.05;t=3.53,P0.01)。【结论】1.慢加急性肝衰竭小鼠的24h累计存活率降低,其肝脏、脾脏、外周血的Breg细胞表达频率及IL-10的表达高于对照组,Breg在慢加急性肝衰竭小鼠疾病进展中起重要作用。2.MSC移植治疗可提高ACLF小鼠生存率,可能与MSC下调Breg数量以及IL-10分泌有关。
[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卤0.70)pg/ml,(21.21卤1.62)Vs(8.23卤1.52)pg/ml,(31.32卤2.95)4.Vs(7.49卤1.22)pg/ml,P鍊煎潎灏忎簬0.001.5.MSC娌荤枟缁勫皬榧，

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