Studies On Burn Healing Enhancement Mechanisms Of UC-MSCs Transplantation With Activiated Ido Expression In Rat Models

Objects: To explore the immunoregulation effect of UC-MSCs(Umbilical cord derived mesenchymal stem cells) with high IDO expression on T lymphocytes by coculture and rodent burn model by transplantation, which may guide an prospective path for burn treatment. Methods:(1) In vitro experiment: Stably cultured UC-MSCs was induced by IFN-γ(Interferon-γ) for IDO expression, which was detected by RT-PCR(Reverse Transcription Polymerase Chain Reaction) and WB(Western Blotting) in mRNA level and protein level respectively. Then induced MSCs, induced MSCs with antagonist1-MT and MSCs were cocultured with T lymphocytes at several ratios for evaluation of immunoregulation effects in vitro.(2) In vivo experiment in rat: Once the rat burn model was constructed, the inflammation markers including WBC, CRP, IFN-γ, TNF-α, IL-6 and IL-10 in burn models were detected longitudinally. Based on the longitudinal variation of inflammation markers in burn model, we made an optimal strategy as transplanting cells24 h later after burn and around the local wounds. Then 240 male SD(sprague-dawley)rat(weight at 200±5g) were randomly grouped as blank control group, saline control group,MSCs group, Induced MSCs group and Induced MSCs +1-MT group according to different transplanted materials. The expression level of WBC, CRP, IFN-γ, TNF-α, IL-6and IL-10 was detected at 0d、1d、2d、3d、5d、7d、9d after burn and the results from different groups were analyzed by ANOVA(analysis of variation). 28 days later after burn,the histological sections were collected from transplanted rat model and taken photos, then the image data was analyzed and compared by software Image J for comparing wound healing rates between test and control groups. Results:(1) The results of RT-PCR and WB showed that UC-MSCs express IDO in a very low level, but after induction, IDO expression significantly increased both in m RNA and protein levels.(2)In the process of coculture, high IDO expression in UC-MSCs caused slower T lymphocytes proliferation;however, the specific antagonist 1-MT could reverse the suppression effect.(3) WBC,CRP, IFN-γ, TNF-α, IL-6 and IL-10 leveled up in about 7 days after burn. Transplantation of induced MSCs and MSCs could reduce WBC, CRP, TNF-α, IL-6, IL-10 peak and induced MSCs group showed more reduction than MSCs, but 1-MT could impede the effects of IDO induced by IFN-γ. Besides, all groups showed no statistical difference in rat IFN-γ expression.(4)14 days after burn, MSCs group, Induced MSCs group and Induced MSCs +1-MT group showed a better healing rate than control group but no difference among themselves. 21 days and 28 days later, the comparison results changed as Induced MSCs group > MSCs group > Induced MSCs+1-MT group > other groups. Conclusion:Induced UC-MSCs with high IDO expression could suppress T lymphocyte proliferation in coculture significantly. After transplantation, UC-MSCs could promote the healing process in rat burn model by reduce several classic inflammation markers. Moreover,when IDO was upregulated in induced UC-MSCs, cells showed better effects for burn recovery. UC-MSCs transplantation could benefit burn treatment in vessel reconstruction,fibroblast accumulation and skin remodeling by immunoregulation, IDO may play an important role during this process.