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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(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,其余各实验组均高于加入前(P<0.05)。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(P<0.05)。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。
Objective To investigate the effect of indoleamine 2,3-dioxygenase (IDO) gene transfection on the rejection of allogeneic composite tissue allograft in rats and its mechanism. Methods The 3rd generation BMSCs from Donor 4 to 6-week-old BN rats were transfected with IDO overexpression lentivirus IDO [Lentiviral green fluorescent protein (GFP)] - Lenti. The target gene was screened for high expression, Active cell line IDO-BMSCs. The expression of IDO mRNA and protein in BMSCs before and after gene modification was detected by RT-PCR and Western blot. The biological activity of IDO was detected by measuring the amount of kynurenine in the culture supernatant. Mixed lymphocyte reaction system in IDO-BMSCs, reactive cells (recipients 4 to 6 weeks old LEWIS rat derived peripheral blood mononuclear cells) and stimulated cells (donor BN rat derived peripheral blood mononuclear cells) mixed culture , The cell ratio was 1: 5: 5, 1:10:10, 1:50:50 and 1: 100: 100 respectively (experimental group 1, 2, 3, 4) , 1 mmol / L IDO specific inhibitor 1-methyl-tryptophan (1-MT) was added. IDO-BMSCs and reactive cells (1: 5) And the reaction cells (1: 1) were cultured as a positive control group, IDO-BMSCs cultured in RPMI1640 medium alone as a blank control group. After cultured for 5 days, the proliferation of T lymphocytes was detected by MTT assay. The model of limb allotransplantation was further established. The GFP-BMSCs (group A), IDO-BMSCs (group B) and saline (group C) were transplanted via the tail vein into the transplantation model to observe the graft survival time and immune rejection reaction. Results After gene modification, the expression of IDO mRNA and protein in BMSCs was significantly increased. Compared with GFP-BMSCs, IDO-BMSCs significantly increased kynurenine content (P <0.05). Before the addition of 1-MT, the proliferation rate of T lymphocytes in experimental groups 1, 2 and 3 increased with the proportion of IDO-BMSCs and reactive cells decreasing gradually, with statistical significance (P <0.05). The experimental group 4 T lymphocyte proliferation rate compared with the positive control group no significant difference (P> 0.05). After addition of 1-MT, except for the proliferation rate of T lymphocyte in experimental group 4, there was no significant difference (P> 0.05) between the addition of T-lymphocyte and the other experimental groups. In vivo, IDO-BMSCs colonized the graft and inhibited the immune rejection. The survival time of group A, B and C were (11.5 ± 0.6), (14.5 ± 0.8) and (9.0 ± 0.3) d, the survival time in group B was significantly longer than that in group A and C, and that in group A was longer than that in group C (P <0.05). Conclusion IDOBMSCs can promote the survival of rat allograft tissue graft by inhibiting the proliferation of T lymphocytes and promoting the implantation of BMSCs into the graft.