白介素1受体拮抗剂壳聚糖纳米颗粒联合骨髓间充质干细胞移植治疗急性肝衰竭的实验研究  

A novel treatment regimen for acute liver failure based on a combination of mesenchymal stem cells transplantation and IL-IRa-ioaded chitosan nanoparticles

作　　者：肖江强[1] 施晓雷[1] 檀家俊[1] 张玲[2] 许茜[2] 丁义涛[1] 

机构地区：[1]南京大学医学院附属鼓楼医院肝胆外科,210008 [2]东南大学公共卫生学院

出　　处：《中华肝脏病杂志》2013年第4期308-314,共7页Chinese Journal of Hepatology

基　　金：国家自然科学基金青年科学基金（30901431）

摘　　要：目的探讨炎症因子白细胞介素1受体拮抗剂（IL-1Ra）与骨髓间充质干细胞（MSCs）移植联合治疗肝功能衰竭的协同效应及其机制。方法采用静电喷射法制备IL-1Ra壳聚糖纳米颗粒，酶联免疫吸附法测定IL-1Ra纳米颗粒体外缓释作用，荧光显微镜和流式细胞术观察纳米颗粒肝细胞靶向性。将IL-1Ra纳米颗粒与MSCs联合治疗急性肝衰竭（ALF）幼猪，具体方法为：D-氨基半乳糖诱导建立猪ALF模型，34头中华实验小型猪随机分为5组，造模对照组经门静脉注射等渗盐水；IL-1Ra组经门静脉注射等渗盐水6h前耳背大静脉推注IL-1Ra；MSCs组经门静脉移植MSCs；IL-1Ra联合MSCs组和IL-1Ra纳米颗粒联合MSCs组先由耳背大静脉分别推注IL-1Ra或IL-1Ra纳米缓释颗粒，再经门静脉移植异体MSCs。移植后4周内定期检测肝功能、炎症因子变化和病理改变，并监测移植细胞体内存活、转化和分淤情况。组间数据比较用独立样本t检验。结果IL-1Ra纳米颗粒在磷酸盐缓冲液中呈现缓慢释放过程；绿色荧光显示乳糖酰基化的纳米颗粒能特异性地靶向到肝细胞。IL-1Ra纳米颗粒联合MSCs组具有显著炎症环境改善作用，与对照组相比，肿瘤坏死因子α在术后1周即明显下降，而IL-6水平在各时间点均明显降低；且IL—IRa纳米颗粒联合MSCs有促进肝功能恢复和肝脏细胞增殖的作用[术后第3周，每个200倍视野下的细胞数为（68．8±9．1）个，与对照组的（21．2±2．5）个相比，t=5．173，P〈0．01]；免疫组织化学及荧光显微镜显示该组MSCs存活数量明显增多；Westernblot结果显示该组分泌肝细胞生长因子及血管内皮生长因子较其余各组明显增多。结论IL-1Ra乳糖酰基壳聚糖纳米颗粒具有良好的肝脏靶向和药物缓释作用，联合MSCs移植具有明显协同作用，通过改善炎症环境和分泌多种细胞因子显著改善D-氨基半乳糖诱导的ALF动�Objective To evaluate whether a combination therapy using allogeneic mesenchymal stem cell （MSC） transplantation and interleukin-1 receptor antagonist （IL-1Ra） chitosan nanoparticles is more robust than MSC transplantation alone for treating acute liver failure and to investigate the mechanisms of the improved therapeutic effect using a swine model system. Methods IL-1Ra-loaded nanoparticles were made of lactosylated chitosan-FITC using the electrostatic spray method and analyzed by enzyme-linked immunosorbent assay. The active live targeting of these nanopaticles were investigated by fluorescence microscope and flow cytometer（FCM）. The combined therapy was given and the Detailed Steps as follows： Chinese experimental mini swine were given D-galactosamine to build models of acute liver failure. Thirty-four pigs were randomly divided into five groups. In the control group（A）,the normal saline was injected into liver via portal veins after 24 h; in IL-1Ra group（B）, IL-1Ra was injected via ear veins 6 h before normal saline; In the MSCs transplantation group （C）, 8 x 107 MSCs were injected into liver via portal veins after 24 h; IL-1Ra together with MSCs transplantation group（D） and nanopaticles group（E） as follows： on the one hand, 8 ~ 107 MSCs were injected into liver via portal veins after 24 h, on the other hand, rhIL-1Ra in group C or IL-1Ra chitosan nanopaticles in group D was injected via ear veins respectively at 6 h before. Liver function, serum inflammation and pathological changes were measured. The fate of MSCs was also observed. Results The profiles in vitro shown that there was a steady-state release after a fast linear release; The live targeting of the lactosylated chitosan-based nanoparticles was achieved by ligand-receptor specificity; The biochemical assay, the serum inflammation lever and pathological changes of the nanopaticles group were all greatly different from the other groups, the hepatocytes grow rate was significantly improved after 1 week; The liver e

关 键 词：肝功能衰竭 急性 间质干细胞移植 壳聚糖 纳米粒子 白细胞介素1受体拮抗剂 

分 类 号：R575.3[医药卫生—消化系统]