成熟树突状细胞在肝脏缺血再灌注中的作用

Effects of mature dendritic cells on mouse liver ischemia/reperfusion injury

目的:探讨成熟树突状细胞(dendritic cells,DCs)在小鼠肝脏缺血再灌注中的作用。方法:收集骨髓源性树突状细胞(bone marrow dendritic cells,BMDCs),经过不同处理(正常肝细胞培养上清或缺氧再氧合原代肝细胞培养上清培养24 h)后,在细胞流式仪上检测细胞成熟相关指标(CD40、CD80、CD86、MHCⅡ);然后将20只健康雄性C57BL/6小鼠随机分成缺血再灌注组(IR)、未经刺激的骨髓源性树突状细胞处理组(IR+NEG-BMDCs)、正常肝细胞上清刺激的骨髓源性树突状细胞处理组(IR+CON-BMDCs)、经缺氧再氧合的原代肝细胞上清刺激成熟骨髓源性树突状细胞处理组(IR+H/R-BMDCs),各组5只。采用70%肝脏缺血再灌注模型(缺血1 h,再灌注6 h)。各组分别于术前1 h给予PBS、NEG-BMDCs、CON-BMDCs、H/R-BMDCs尾静脉注射。酶联免疫吸附实验(ELISA法)分别测定血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、白介素-10(IL-10)、白介素-17(IL-17)水平。通过光镜观察组织HE染色改变。反转录酶-聚合酶链式反应(RT-PCR)检测肝组织转化生长因子β(TGF-β)、叉头框蛋白P3(FOXP3)、IL-10、IL-17水平。结果:H/R-BMDCs处理组肝酶水平明显低于其他3组(P<0.05),形态学分析及Suzuki评分明显优于其他3组。H/R-BMDCs处理组血清及肝组织中IL-10、肝组织中TGF-β、FOXP3表达水平明显高于其他组,IL-17低于其他组(P<0.05)。结论:H/R-BMDCs预处理可以通过调节调节性T细胞(Treg)和辅助性T细胞17(Th17)的平衡来减轻小鼠肝脏缺血再灌注损伤。

Objective：To investigate effects of mature dendritic cells（DCs）on mouse liver ischemia/reperfusion（I/R）injury. Methods：After different treatment of bone marrow-derived dendritic cells collected,staining was performed according to the reagent protocol,and the relevant indexes were detected on the cell flow meter（CD40,CD80,CD86,and MHCⅡ）. A total of 20 healthy male C57BL/6 mice were randomly divided into four main experimental groups（n=5 each）,including the ischemia/reperfusion（I/R）group,the NEG-BMDCs pretreatment group,the CON-BMDCs pretreatment group,and the H/R-BMDCs pretreatment group. We chose a nonfatal model of 70% liver I/R（treated with 1 h ischemia,and then 6 h reperfusion）. The mice of I/R group were injected with PBS,the NEG-BMDCs pretreatment group with NEG-BMDCs,the CON-BMDCs pretreatment group with CON-BMDCs,the H/R-BMDCs pretreatment group with H/R-BMDCs,at 1 h before operation. The levels of alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,interleukin-10（IL-10）,interleukin-17（IL-17）in serum were detected by enzyme-linked immunosorbent assay（ELISA）. The mRNA expression of TGF-β,FOXP3,IL-10,IL-17 were examined by reverse transcription-polymerase chain reaction（RT-PCR）. Histological haema（HE）stained sections were histopathologically examined using light microscopy. Results：The liver enzyme level were significantly decreased in the H/R-BMDCs pretreatment group,compared to those in the other groups（P 〈 0.05）. Morphometric analysis and Suzuki’s scores showed that H/R BMDCs improved liver ischemia/reperfusion injury（IRI）to a greater extent than BMDCs from the negative and control groups. The expressions of IL-10 of liver tissue blood serum and liver tissue were upregulated and the expressions of TGF-β and FOXP3 of liver tissue were upregulated in the H/R-BMDCs pretreatment group,while the expression of IL-17 was downregulated in the H/R-BMDCs pretreatment group. Conclusion：Pretreatment with H/R-BMDCs protects mouse from I/R injury by modulating the balance between Treg and Th17 cells.