阻断Kupffer细胞IRE1-XBP1通路对大鼠肝移植排斥反应的影响  

Effects of inhibiting IRE1-XBP1 pathway in Kupffer cells in rats after liver transplant rejection

作　　者：王润芝[1] 吴皓[1] 刘一鸣[1] 曹丁[1] 吴涯昆 李金政[1] 龚建平[1] 

机构地区：[1]重庆医科大学附属第二医院肝胆外科,重庆400010

出　　处：《第三军医大学学报》2016年第22期2431-2437,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81200329;81300364)~~

摘　　要：目的探讨阻断Kupffer细胞IRE-XBP1通路对大鼠原位肝移植排斥反应的作用。方法术前用Gd Cl3处理或未处理建立大鼠原位肝移植急性排斥反应模型,未处理大鼠随机数字法分为XBP1-shRNA组(A组),Scrambled-shRNA组(B组),PBS组(C组);处理大鼠随机分为Gd Cl3+XBP1-shRNA组(D组),Gd Cl3+Scrambled-shRNA组(E组),Gd Cl3+PBS组(F组)。未处理组术后检测血清ALT、AST、IFN-γ、IL-10、IL-17的表达水平;RT-PCR检测T-bet、RORγt、IFN-γ、IL-17及IL-10mRNA水平;Western blot检测CD86、CD206、IFN-γ、IL-17及IL-10蛋白表达水平,HE染色观察肝组织结构,根据Banff方案进行RAI评分,各组剩余大鼠观察术后生存率。处理组术后检测IFN-γ、IL-17及IL-10 mRNA以及蛋白表达水平,HE染色观察肝组织结构。结果在未处理组中,与B、C组比较,A组各时间点肝功能指标ALT、AST明显下降(P<0.05),A组血清表达IFN-γ、IL-10明显受到抑制(P<0.05),IL-10的表达水平则呈明显上升趋势(P<0.05),与RT-PCR和Western blot结果趋势相符合,另外A组CD86蛋白表明显下降(P<0.05),而表达CD206上升(P<0.05),T-bet/RORγt mRNA相对表达量也明显下降(P<0.05),B、C组上述指标与A组对比则呈相反趋势,而且B、C组之间比较差异无统计学意义(P>0.05);A组RAI评分(3.83±0.14),明显低于B、C组RAI评分(9.13±0.20)、(8.95±0.26)(P<0.05),并且A组大鼠的生存时间明显高于B、C组大鼠(P<0.05)。在处理组中,D、E和F组肝脏局部IFN-γ、IL-17、IL-10 mRNA和蛋白表达情况以及病理组织AcR程度比较无统计学上的差异(P>0.05)。结论阻断IRE1-XBP1通路促进了KCs的M1型极化,引起Th1/Th17向Th2/Treg的免疫偏移,在一定程度上减轻了移植肝脏急性排斥反应的程度。Objective To investigate the effects of inhibiting inositol requiring 1-X-Box binding protein 1 （IRE1-XBP1） pathway in Kupffer cells （KCs） on liver transplant rejection in rats. Methods Orthotopic liver transplantation in rats was established with or without GdC13 treatment pre-operation. In untreated group, the rats were divided randomly into 3 subgroups, that is, XBPI-shRNA group （A） , Scrambled-shRNA group （ B）, and PBS group （ C ）. Likewise, the treated group was sub-divided into GdC13 ± XBPI-shRNA group （D）, GdC13 ± Serambled-shRNA group （E）, and GdCI3 ± PBS group （F）. In untreated group, the serum levels of alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, interferon-γ （IFN-3,）, interleukin-10 （IL-10） and IL-17 were examined at different time points after operation. Liver tissues were excised for hematoxylin and eosin （HE） staining, mRNA expression levels of transcriptional factors （ T-bet and RORγ/t） and eytokines （IFN-γ, IL-17 and IL-10） quantified by real-time PCR （RT-PCR）, and protein levels of CD86, CD206, IFN-γ, IL-17 and IL-10 by Western blotting. Banff schema was used for grading theacute allograft rejection with rejection activity index （RAI）. The survival rate was assessed by Kaplan-Meier curve analysis. In the treated group, the mRNA and the protein expression of IFN-γ, IL-17 and IL-10 was also tested. Results In the untreated group, the serum levels of ALT and AST at different time points were significantly lower in group A compared with groups B and C （P 〈 0. 05 ）. In group A, IFN-γand IL-17 were significantly decreased in serum while IL-10 was significantly increased （ P 〈 0. 05 ）. There were consistent with the results of RT-PCR and western blotting. In addition, the relative mRNA expression of T-bet/RORγt was decreased, protein expression of CD86 decreased and CD206 increased, which were all statistically significant （P 〈 0. 05）. In contrast, groups B and C showed inverse trend wit

关 键 词：肌醇酶1 X盒蛋白连接蛋白1 KUPFFER细胞 肝移植 急性排斥反应 

分 类 号：R392.4[医药卫生—免疫学] R617[医药卫生—基础医学]