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

发布时间：2019-05-07 05:51

【摘要】：目的探讨吲哚胺2,3-双加氧酶(indoleamine 2,3-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(green fl uorescent protein,GFP)]-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、Western blot检测基因修饰前后BMSCs中IDO mRNA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1 mmol/L IDO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RPMI1640培养基中单独培养作为空白对照组。于培养5 d,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IDO mRNA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(P0.05)。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(P0.05);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(P0.05)。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(P0.05)外,其余各实验组均高于加入前(P0.05)。体内输入后,IDO-BMSCs可在移植物内定植,抑制免疫排斥反应的发生;A、B、C组移植物存活时间分别为(11.5±0.6)、(14.5±0.8)、(9.0±0.3)d,B组存活时间明显长于A、C组,A组长于C组(P0.05)。结论 IDOBMSCs可促进大鼠同种异体复合组织移植物的成活,其机制与抑制T淋巴细胞增殖、促进BMSCs向移植物内定植有关。
[Abstract]:Aim to investigate the effect of indoleamine 2, 3-dioxygenase (indoleamine-2) gene transfection on the immune rejection of allogeneic composite tissue transplantation in rats and its mechanism. Methods BMSCs-modified rats were transfected with indoleamine-2, 3-dioxygenase (IDO) gene. Methods IDO overexpressed lentivirus IDO [green fluorescent protein (green fl uorescent protein,GFP]-Lenti was transfected into the third generation BMSCs, derived from 4-6-week-old BN rats to screen the target gene of IDO-BMSCs. cell line with high expression and biological activity. RT-PCR,Western blot was used to detect the expression of IDO mRNA and protein in BMSCs before and after gene modification, and the biological activity of IDO was detected by measuring the production of urine in cultured canine supernatant. In the mixed lymphocyte reaction system, IDO-BMSCs, reactive cells (peripheral blood mononuclear cells from 4-week-old LEWIS rats) and stimulating cells (peripheral blood mononuclear cells from donor BN rats) were co-cultured. The proportion of cells was 1-5-5, 1-10-10, 1-50-50 and 1-100-100, respectively (experimental group 1, 2, 3, 4). IDO blocking pore was set up in each reaction system, and 1-mmol/L IDO specific inhibitor 1-methyl-tryptophan (1-MMT) was added to each reaction system in the presence of 1-methyltryptophan (1-methyltryptophan). IDO-BMSCs and reactive cells (1:5) were used as negative control group, stimulating cells and reactive cells (1:1) were cultured as positive control group, and IDO-BMSCs was cultured in RPMI1640 medium as blank control group. After 5 days of culture, the proliferation of T lymphocytes was detected by MTT method. GFP-BMSCs (group A), IDO-BMSCs (group B) and saline (group C) were injected into the model by tail vein to observe the survival time and immune rejection. Results the expression of IDO mRNA and protein in BMSCs was significantly increased after gene modification, and the content of urine in cultured supernatant was significantly increased by IDO-BMSCs than that of GFP-BMSCs (P0.05). Before adding 1-MT, the proliferation rate of T lymphocytes in experiment group 1, 2 and 3 increased with the decrease of the ratio of IDO-BMSCs and reactive cells, and the difference between groups was statistically significant (P0.05). There was no significant difference in T lymphocyte proliferation rate between experimental group 4 and positive control group (P0.05). After the addition of 1-MT, there was no significant difference in T lymphocyte proliferation rate between experimental group 4 and pre-addition group (P0.05), and all the other experimental groups were higher than those before addition (P0.05). After administration in vivo, IDO-BMSCs could be implanted in the graft to inhibit the occurrence of immune rejection. The graft survival time in group A, B and C was (11.5 卤0.6), () 14.5 卤0.8), (9.0 卤0.3 days, respectively. The survival time in group B was significantly longer than that in group A, and in group C, the survival time of group A was significantly longer than that in group C (P0.05). Conclusion IDOBMSCs can promote the survival of allogeneic composite tissue graft in rats, and its mechanism is related to the inhibition of T lymphocyte proliferation and the promotion of BMSCs implantation into the graft.
【作者单位】：第四军医大学西京医院全军烧伤中心;
【基金】：国家自然科学基金青年项目(81301632)~~
【分类号】：R392

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