| Objective The purpose of this study was to investigate the effects of CD200Genetransfected Human Placenta-Derived Mesenchymal Stem Cells (HPMSCs) on ImmuneRejection in Mouse Allogenic Skin Transplantation. The results may provide new clues forunderstanding the immunoregulatory mechanism of CD200Gene. In order to study CD200gene-mediated immunoregulatory pathway in animal models, we used HPMSCs transfectedwith mouse CD200Gene to replaced CD200positive MSCs.Methods In order to establish HPMSCs lines which stably express mouse CD200gene,we constructed the recombinant adenovirus vector containing mouse CD200gene, then theadenovirus was used to infect HPMSCs. The infected cells were digested into single cellsuspension after they reached60~70%confluence. The Vr:CD1(ICR) mice were used as skindonor and C57BL/6mice as recipient for allogenic skin transplantation．Forty recipient micewere divided into four groups:①Blank control group1: autograft;②control group2:allogenic graft+PBS iv. Injection;③control group3: allogeneic graft+PMSCs iv. Injection;④experimental group: allogeneic graft+CD200-transfected PMSCs iv. Injection.Thesurvival times and pathologic changes of skin grafts were observed by macro-and microscopewith HE staining. Seven days after skin transplantation, blood leukocyte counting, abdominalfluid macrophage activation, and cytokine levels in blood and spleen were detected by cellstaining and morphology analysis, ELISA and RT-PCR, respectively． Results (1) Successful construction of recombinant adenovirus expressing mouse CD200gene was demonstrated through PCR, restriction enzyme digestion, DNA sequencing andGFP expression of GFP.(2) After infection of HPMSCs with CD200expressing adenovirusvectors, gene-transfected HPMSC cell lines stably expressing mouse CD200gene wereestablished. The expression of mouse CD200by HPMSCs was verified by fluorescencemicroscope and real-time PCR.(3) The effects of CD200on survival time of the skin graftswere evaluated. Comparing with group B (allogeneic graft+PBS iv.injection), the survivaltime of the skin grafts in group C (allogeneic graft+HPMSCs iv.injection) was significantlylonger(p＜0.001); Comparing with group B and group C, the survival time of the skin graftsin group D (allogeneic graft+CD200-transfected HPMSCs iv. Injection) was significantlylonger(p＜0.001, p＜0.001). HE staining the graft tissues showed that the inflammation cellinfiltration was significantly decrease in group D in comparison with group B. Comparingwith group A and group B, a significant decrease in blood leukocyte number was observedin group D (p＜0.05, p＜0.05) on day7after transplantation. The numbers of activatedmacrophages in group C and group D were significantly higher than that in groups A and B (p＜0.05, p＜0.05, p＜0.05, p＜0.05). ELISA results showed that the concentrations of IL-4were significantly reduced in group C and group D comparing with group A (p＜0.05, p＜0.05); IL-17and IFN-γ were significantly lower in group C (p＜0.01, p＜0.01) and group D(p＜0.001, p＜0.001) than that group B, RT-PCR results showed that a decrease of IL-4,IL-17and IFN-γ was detected in group C (p＜0.05，p＜0.01，p＜0.01) and group D (p＜0.01，p＜0.01, p＜0.01) when compared with group B. Comparing with group A, the concentrationof IFN-γ was significantly lower in group C and group D (p＜0.01, p＜0.05).Conclusions A recombinant adenovirus vector expressing mouse CD200gene(rAd-4-mCD200) was constructed successfully. After transfection with this vector, mouseCD200gene was stably and efficiently expressed in HPMSCs. Both HPMSCs and CD200 transfected HPMSCs protected allogenic skin grafts from immune rejection, but the HPMSCsexpressing mouse CD200prolonged the survival time of allogenic the skin grafts more thanuntransfected HPMSCs, demonstrating a immunosuppressive role exerted by CD200．BothHPMSCs and CD200infected HPMSCs also suppressed acute serological immune responsesto allogenic skin transplantation. The possible mechanism underlying CD200mediatedimmunosuppression may be partially related to its inhibitory effects on the secretion of IL-4,IL-17and IFN-γ by T cells. |
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