吲哚胺2,3-双加氧酶基因修饰BMSCs在大鼠同种异体复合组织移植免疫排斥反应中的作用研究  

EFFECTS OF INDOLEAMINE 2,3-DIOXYGENASE GENE MODIFIED BONE MARROW MESENCHYMAL STEM CELLS IN RAT COMPOSITE TISSUE ALLOGRAFT REJECTION

作　　者：韩夫[1] 王耀军[1] 王耘川[1] 贾艳慧[1] 杨龙龙[1] 贾文斌[1] 方小兵[1] 白晓智[1] 胡大海[1] 

机构地区：[1]第四军医大学西京医院全军烧伤中心,西安710032

出　　处：《中国修复重建外科杂志》2014年第6期745-751,共7页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金青年项目(81301632)~~

摘　　要：目的探讨吲哚胺2，3-双加氧酶（indoleamine2，3-dioxygenase，IDO）基因转染修饰大鼠BMSCs对同种异体复合组织移植免疫排斥反应的影响及其机制。方法以IDO过表达慢病毒IDO[绿色荧光蛋白（greenfluorescentprotein，GFP）1-Lenti转染供体4～6周龄BN大鼠来源的第3代BMSCs，筛选目的基因高表达、具有生物活性的细胞株IDO-BMSCs。行RT-PCR、Westernblot检测基因修饰前后BMSCs中IDOmRNA及蛋白表达，通过测量培养上清犬尿氨酸生成量检测IDO生物学活性。在混合淋巴细胞反应体系中以IDO-BMSCs、反应细胞（受体4～6周龄LEWIS大鼠来源外周血单个核细胞）和刺激细胞（供体BN大鼠来源外周血单个核细胞）混合培养，细胞比例分别为1：5：5、1：10：10、1：50：50及1：100：100（实验组1、2、3、4）：每个反应体系另设IDO阻断孔，加入1mmol／LIDO特异性抑制剂1-甲基色氨酸（1-methyl．tryptophan，1-MT）；以IDO-BMSCs与反应细胞（1：5）培养为阴性对照组，刺激细胞与反应细胞（1：1）培养为阳性对照组，IDO-BMSCs在RPMll640培养基中单独培养作为空白对照组。于培养5d，采用MTT法检测T淋巴细胞增殖情况。进一步制备大鼠同种异体肢体移植模型，将GFP-BMSCs（A组）、IDO-BMSCs（B组）及生理盐水（C组）分别经尾静脉输入移植模型，观察移植物存活时间及免疫排斥反应。结果基因修饰后，BMSCs中IDOmRNA及蛋白表达显著提高。IDO-BMSCs较GFP-BMSCs可明显提高培养上清中犬尿氨酸含量（P〈0．05）。加入1-MT前，实验组1、2、3的T淋巴细胞增殖率随IDO-BMSCs与反应细胞比例逐渐减少而不断增加，组间比较差异均有统计学意义（P〈0．05）；实验组4的T淋巴细胞增殖率与阳性对照组比较差异无统计学意义（P〉0．05）。加入1-MT后，除实验组4的T淋巴细胞增殖率与加入前比较差异无统计学意义（P〉0．05）外，其余各实验组均�Objective To evaluate the effects and mechanism of indoleamine 2, 3-dioxygenase （IDO） modified rat bone marrow mesenchymal stem cells （BMSCs） in composite tissue allograft rejection. Methods BMSCs isolated from Brown Norway （BN） rats （aged, 4-6 weeks） were infected by IDO [green fluorescent protein （GFP）]-lentivirus. The high expression target gene and biological activity cell line （IDO-BMSCs） were screened. IDO mRNA and protein expressions were detected by RT-PCR and Western blot. The biological activity of IDO in supernatant was detected by measuring the amount of kynurenine generation. In mixed lymphocyte reaction system, different numbers of IDO-BMSCs mixed with responding cells （peripheral blood mononuclear cell isolated from 4-6-week-old LEWIS rats, as recipient） and stimulating cells （peripheral blood mononuclear cell isolated from BN rats, as donor）, with the cells ratios of 1 ： 5 ： 5, 1 ： 10 ： 10, 1 ： 50 ： 50, and 1 ： 100 ： 100 （as experimental groups 1, 2, 3, and 4, respectively）. Each reaction system was blocked by 1 mmol/L 1-methyl-tryptophan （1-MT） （IDO specific inhibitor）. IDO-BMSCs mixed with responding cells （1 ： 5） as the negative control group, responding cells mixed with stimulating cells （1 ： 1） as positive control group; and IDO-BMSCs were cultured in RPMI1640 medium alone as blank control group. MTT assay was used to detect the T lymphocytes proliferation at 5 days. Furthermore, GFP-BMSCs （group A）, IDO-BMSCs （group B）, and normal saline （group C） were infused via the tail vein of allogeneic limb transplantation rats, and graft survival time and rejection were observed in each group. Results The IDO expression of BMSCs after genetic modification was higher than that before genetic modification. IDO-BMSCs could significantly improved kynurenine concentration in culture medium supernatant when compared with GFP-BMSCs （P 〈 0.05）. Before adding 1-MT, with the ratio of IDO-BMSCs to responding cells decreased

关 键 词：BMSCS 吲哚胺2 3-双加氧酶 T淋巴细胞 同种异体复合组织移植 免疫排斥反应 大鼠 

分 类 号：R392[医药卫生—免疫学]