大鼠肝移植急性排斥反应的淋巴细胞蛋白质组学研究

Proteomics analysis of lymphocyte involving in acute rejection after liver transplantation within rats

目的利用差异蛋白质学寻找肝移植急性免疫排斥反应相关功能蛋白。方法选取SD大鼠与Wistar大鼠，建立大鼠同种异体肝移植的动物模型（急性排斥组）和大鼠同基因移植的动物模型（对照组）；使用组织化学方法对移植肝脏进行形态学观察；利用ELISA检测受体血清细胞因子；通过双向凝胶电泳分离急性排斥组和对照组肝移植后受体大鼠脾脏的淋巴细胞蛋白质，通过软件比较两组蛋白质的质纹图谱，进行差异点分析；选取差异点用基质辅助激光解析-飞行时间质谱进行蛋白鉴定；选取部分鉴定蛋白通过Westernblot法进行检测，验证蛋白质组学的分析结果。结果ELISA检测结果表明，移植术后3d同种异体肝移植大鼠血清中IL-2和IFN-γ的表达上调；急性排斥组肝组织HE染色切片证实均有Ⅱa～Ⅱb级（Banff标准）排斥反应表现；差异蛋白质组学分析共鉴定出25个淋巴细胞蛋白质在急性排斥反应中的表达量发生了改变，其中13个蛋白点表达上调，12个蛋白点表达下调；Westernblot法验证结果显示，其中的2个相关蛋白（β-actin和碳酸酐酶）在急性排斥反应中的表达量变化与差异蛋白质组学分析结果一致。结论本实验筛选出25个大鼠肝移植急性排斥反应的相关功能蛋白，其中的β-actin和碳酸酐酶在排斥反应中有重要功能，为进一步系统研究这一免疫学现象奠定了基础。

Objective To screen specific functional proteins from lymphocyte involved in acute rejection using differential proteomics research. Methods Two groups of rat liver transplantation models were established（ isograft as control and allograft as acute rejection groups） by transplantation within Wistar rats, and between Wistar and SD. Morphology study were performed by histocbemistry tech, followed by serum cytokine detection with ELISA. With 2-dimensional electrophoresis, proteomes of lymphocyte from the rats of different groups were separated and 2 proteome profiles were established. Comparing with the 2 profiles, 25 spots were selected and picked for in gel digestion, followed for analysis by matrix assisted laser desorption ionization （MALDI）-time of fly （TOF）/TOF MS. Two of the proteins were detected with Western blot to verify the changing profiles. Results The results of morphology analysis and detection of cytokines （ IL-2 and IFN-γ） indicate that the animal models were established successfully and acute rejection happened after transplantation for 3 days. Twenty-five differential proteins were found out to be associated with acute rejection,among which 13 proteins were upregulated and 12 downregulated. The expression alterations of 2 proteins （β-actin and carbonic anhydrase） are consistent with proteomics analysis results showing in Western blot. Conclusions Twenty-five specific proteins exploiting mechanism of acute rejection are screened out, including IL-2 and carbonic anhydrase ,which maybe benefit for the further works.