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

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.

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 RPM11640 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.

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, T lymphocytes proliferation rate increased in experimental groups 1, 2, and 3, showing significant differences between groups (P < 0.05); there was no significant difference between experimental group 4 and the positive control group (F > 0.05). After adding 1-MT, T lymphocytes proliferation rate was significantly higher than that before adding 1-MT in the other experimental groups (P < 0.05) except experimental group 4 (F > 0.05). In vivo, IDO-BMSCs could promote colonization in allograft, inhibit transplantation rejection, and prolong survival time of composite tissue allograft; the survival time of composite tissue allograft was (11.5 ± 0.6) days in group A, (14.5 ± 0.8) days in group B, and (9.0 ± 0.3) days in group C, and it was significantly longer in group B than in groups A and C, and in group A than in group C (P < 0.05).

JDO-BMSCs can promote the survival of allogeneic composite tissue grafts in rats, and its mechanism may involve in inhibition of T lymphocytes proliferation and promotion their own colonization in allograft.