文章摘要
姚传霞,王怡雯,龚丹丹,李丽莉,马 艳,匡 衡,周婷婷,汪茂荣.人源抗TLR4抗体IgG2对对乙酰氨基酚诱导小鼠急性肝损伤的保护作用[J].南京医科大学学报,2020,(5):645~651
人源抗TLR4抗体IgG2对对乙酰氨基酚诱导小鼠急性肝损伤的保护作用
Protection effects of hTLR4 IgG2 on acetaminophen⁃induced acute liver injury in mice
投稿时间:2019-10-11  
DOI:10.7655/NYDXBNS20200506
中文关键词: 人源抗TLR4抗体IgG2  对乙酰氨基酚  炎症因子  细胞凋亡
英文关键词: hTLR4 IgG2  acetaminophen  inflammation factor  apoptosis
基金项目:国家“十二五”规划项目(2013ZX09J13110?05B);江苏省社会发展项目资助(BE2018617);中国重点技术重点项目基金(2018YFC1200603)
作者单位
姚传霞 安徽医科大学解放军八一临床学院肝病中心安徽 合肥 230000 
王怡雯 东部战区疾病预防控制中心江苏 南京 210002 
龚丹丹 南京医科大学附属妇产医院妇产科江苏 南京 210004 
李丽莉 安徽医科大学解放军八一临床学院肝病中心安徽 合肥 230000 
马 艳 南京中医药大学附属解放军八一医院肝病中心江苏 南京 210002 
匡 衡 安徽医科大学解放军八一临床学院肝病中心安徽 合肥 230000 
周婷婷 东部战区疾病预防控制中心江苏 南京 210002 
汪茂荣 东部战区总医院秦淮医疗区肝病中心江苏 南京 210002 
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中文摘要:
      目的:研究人源抗TLR4 抗体IgG2(human?TLR4 IgG2,hTLR4 IgG2)对对乙酰氨基酚(acetaminophen,APAP)诱导肝损伤的保护效应,探讨其在药物性肝损伤中的保护作用。方法:以人源抗TLR4 抗体Fab基因为模板,扩增其可变区基因,构建人源抗TLR4抗体IgG2真核表达载体,转染CHO?S细胞,筛选稳定表达细胞株,收集细胞上清,Protein G柱纯化抗TLR4抗体IgG2。将18只C57BL/6J 小鼠随机分成3组:生理盐水组、APAP(600 mg/kg)组和APAP +hTLR4 IgG2(5 mg/kg)组,统计小鼠腹腔注射APAP后 24 h的存活率;重复上述实验分组,检测小鼠腹腔注射APAP 8 h后,血清中丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate transaminase,AST)、白细胞介素?1(interleukin?1,IL?1)、白细胞介素?6(interleukin?6,IL?6)和肿瘤坏死因子?α(tumor necrosis factor?α,TNF?α)的表达水平,取肝脏做病理分析并使用Western blot检测凋亡蛋白的表达。结果:成功构建并表达纯化人源抗TLR4抗体IgG2;ELISA结果显示,抗体效价为1∶204 800。与APAP组相比,APAP+hTLR4 IgG2组小鼠的24 h存活率显著增加(P < 0.05);血清AST、ALT以及炎性细胞因子的表达水平显著降低,具有统计学意义(P < 0.05);病理分析结果显示,与模型组相比,人源抗TLR4抗体IgG2治疗组的小鼠肝组织炎性细胞浸润、充血和坏死等症状明显改善;Western blot结果显示凋亡相关蛋白的表达减少。结论:人源抗TLR4抗体IgG2能够抑制炎症因子的表达及细胞凋亡,对APAP诱导的小鼠肝损伤有显著的保护作用。
英文摘要:
      Objective:This study aims to ohserve the protective effect of human?TLR4 IgG2(hTLR4 IgG2)on acetaminophen(APAP) induced liver injury,and to investigate the protective mechanism of hTLR4 IgG2 in drug induced liver injury. Methods:The hTLR4 Fab plasmid in our laboratory were used as templates to amplify the variable region genes,and the hTLR4 IgG2 eukaryotic expression vector was constructed and transfected into CHO?S cells. The stable transfectants strains and culture supernatants were collected. Finally,hTLR4 IgG2 was purifieded using a protein G column. Total 18 C57BL/6J mice were randomly divided into three groups:saline group,APAP(600 mg/kg)and the APAP+hTLR4 IgG2(5 mg/kg)group. The survival rate of mice after 24 hours intraperitoneal injection with APAP was compared. Repeat the above experimental grouping and modeling methods were repeated. Aspartate transaminase(AST),alanine aminotransferase(ALT),interleukin?1(IL?1),interleukin?6(IL?6) and tumor necrosis factor?α(TNF?α) in serum were also qualified,the liver was taken for pathological section and the expression of hepatic apoptosis protein was analyzed by Western blot after 8 hours intraperitoneal injection with APAP. Results:The hTLR4 IgG2 was successfully constructed and purified,and the results of ELISA showed that the antibody titer was 1∶204 800. Compared with the APAP group,the 24?hour survival rate of mice in the APAP+hTLR4 IgG2 group was significantly increased(P < 0.05);the expression levels of AST,ALT and inflammatory cytokines in serum were significantly decreased(P < 0.05);pathological detection results showed that the inflammatory cell infiltration,hyperemia and necrosis of liver tissue were significantly improved in APAP+hTLR4 IgG2 group,and the expression of apoptotic protein was decreased in the APAP+hTLR4 IgG2 group by Western blot. Conclusion:hTLR4 IgG2 can effectively inhibit the expression of inflammatory factors and apoptosis,and protect liver against APAP?induced injury in mice.
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