人外周血单个核细胞分泌细胞因子对α-1,3-半乳糖基转移酶基因敲除猪肝细胞的作用  

The effects of cytokines secreted by human peripheral blood mononuclear cell on α-1,3-galactosyltransferase knockout pig hepatocytes

作　　者：王权成 吴文龙 张玄 窦科峰 陶开山 Wang Quancheng;Wu Wenlong;Zhang Xuan;Dou Kefeng;Tao Kaishan(Department of Hepatobiliary Surgery,Xijing Hospital,the Air Force Medical University,Xi′an 710032,China)

机构地区：[1]空军军医大学西京医院肝胆外科,西安710032

出　　处：《中华移植杂志（电子版）》2020年第3期182-187,共6页Chinese Journal of Transplantation(Electronic Edition)

基　　金：国家自然科学基金(81670593,81870446,81671838)。

摘　　要：目的研究异种肝细胞或肝移植中,活化的人外周血单个核细胞(h-PBMC)分泌的细胞因子对α-1,3-半乳糖基转移酶基因敲除猪肝细胞(GalT-KO-hep)的作用。方法利用永生化GalT-KO-hep,与经脂多糖+聚肌胞苷酸激活的h-PBMC共培养。生化分析检测共培养上清中白蛋白、ALT、AST和尿素水平,采用流式细胞术检测GalT-KO-hep凋亡,采用蛋白质印迹法检测GalT-KO-hep中Caspase-3剪切体表达以及c-Jun氨基末端激酶(JNK)、核因子κB(NF-κB)、P38丝裂原活化蛋白激酶(MAPK)、细胞外调节蛋白激酶(ERK)、信号传导与活化转录因子3(STAT3)和蛋白激酶B(AKT)通路相关分子的表达变化。采用方差分析比较不同刺激时间h-PBMC分泌的细胞因子mRNA相对表达量、共培养模型不同时间点上清液生化指标含量、GalT-KO-hep中Caspase 3剪切体表达量和细胞凋亡率,进一步两两比较采用LSD法。P<0.05为差异有统计学意义。结果 GalT-KO-hep与活化h-PBMC共培养6 h组上清液中ALT含量高于共培养24 h和48 h组,差异均有统计学意义(P均<0.05);共培养48 h组AST含量高于共培养6 h和24 h组(P均<0.05);共培养24 h和48 h组尿素含量均高于共培养6 h组(P均<0.05)。GalT-KO-hep与活化h-PBMC共培养24 h和48 h后,细胞中Caspase 3剪切体表达增加,细胞凋亡程度逐渐增加。与活化h-PBMC共培养0.5、1、6和12 h后,GalT-KO-hep磷酸化蛋白p-STAT3、p-JNK、p-IκBα、p-P38 MAPK、p-AKT和p-ERK(1/2)均被不同程度激活。结论 h-PBMC释放的细胞因子可诱导GalT-KO-hep损伤和凋亡,并促进炎症相关分子通路的激活。Objective To investigate the effect of cytokines secreted by activated human peripheral blood mononuclear cell(h-PBMC) on α-1,3-galactosyltransferase knockout pig hepatocytes(GalT-KO-hep) in xenogenic hepatocytes or liver transplantation. Methods LPS, poly(I∶C)-activated h-PBMC were co-cultured with immortalized GalT-KO-hep. Albumin, ALT, AST and urea in co-culture supernatant were detected by biochemical analysis. Flow cytometry was applied to detect the GalT-KO-hep apoptosis. Western blot was used to analysis the expression of cleaved caspase-3 and the proteins related to the signal transduction pathways of c-Jun N-terminal kinase(JNK), nuclear factor kappa-B, p38 mitogen-activated protein kinase(p38 MAPK), extracellular regulated protein kinases(ERK), signal transducer and activator of transcription 3(STAT3) and protein kinase B(AKT). The mRNA levels of cytokines of activated h-PBMC at different point of time, supernate biochemical indexes of co-culture model at different point of time, expression of cleaved caspase-3 and cell apoptosis of GalT-KO-hep were compared by analysis of variance. Further pairwise comparison was performed with LSD method. P <0.05 was considered statistically significant. Results The ALT levels in co-culture supernatant at the time point of 6 hours after cocultivation were higher than 24, 48 hours(P all <0.05). The AST levels in co-culture supernatant at the time point of 48 hours after cocultivation were higher than 6, 24 hours(P all <0.05). The urea levels in co-culture supernatant at the time point of 24, 48 hours after cocultivation were higher than 6 hours(P all <0.05). The expression of cleaved caspase-3 and cell apoptosis in GalT-KO-hep increased 24, 48 hours later after cocultivation;and the expression of p-STAT3, p-JNK, p-IκBα, p-P38 MAPK, p-AKT and p-ERK(1/2) in GalT-KO-hep all increased 0.5, 1, 6, 12 hours later after cocultivation. Conclusions The cytokines released by h-PBMC can induce cell injury and apoptosis of GalT-KO-hep and promote the activation of inflammatory

关 键 词：α-1 3-半乳糖基转移酶基因敲除猪肝细胞 异种肝移植 免疫排斥 外周血单个核细胞 

分 类 号：R657.3[医药卫生—外科学]