Effects Of Human Umbilical Cord Blood-derived Cells Transfected With Ad-Cx43-GFP On The Regulation Of Human Peripheral Blood Mononuclear Cell In Vitro

Objectives:The human umbilical cord blood-derived stromal cells (hUBDSCs) have been show toposses immunomodulatory functions and proposed as a tool for managing or preventinggraft-versus-host disease（GVHD）as well as promoting clinical transplantation tolerance.Despite a lot of work done in this field, there is still much to be investigated and learnedabout the functional roles of hUCBDSCs in the immune system. Connexin (Cx) and gapjunction (GJ)-mediated intercellular communication have been shown to pariticipate in keyimmunological processes, such as Ig secretion and cytokine production, transendthelialmigration of leukocytes, regulatory T cell mediated suppression through the transfer ofcAMP and so on. Cx43is among the most widely distributed and has been shown to be thepredominant Cx43expressed in the bone marrow, thymus, spleen, and other lymphoidtissues. In our previous reports, we confirmed that Cx43was also the predominant connexinin hUCBDSCs. Thus, we speculated that the immunomodulatory functions of hUCBDSCswere also promoted by enhancing the expression of Cx43. We preliminary discussioned thishypothesis by investigating the capacity of the hUCBDSCs which transfected withAd-Cx43-GFP or Ad-GFP to modulate the cytokines secretion(IL-4and IFN-γ),proliferation, apoptosis and cell cycle of lymphocytes.We also analyzed wether regulatoryT cells could be modulated by hUCBDSCs in co-culture.Methods:1. The two step separation procedure of6%gelatin followed by Percoll was used toseparate the mononuclear cells from human umbilical cord blood, and modified Dexterculture system was used to cultured hUCBDSCs. The human peripheral blood lymphocytewere prepared from leucopheresis packs by centrigugation on a Ficoll Hypaque densitygradient. The growth of hUCBDSCs and lymphocytes were observed with invertmicroscope.2. The expression of report gene GFP and the rate of transfection were detected by fluorescent microscopy in hUCBDSCs transfected with recombinant adenovirusAd-Gx43-GFP. After transfected48hours, RT-PCR, Western blot and immunofluorescencewere used to detect the mRNA and protein expressions of Cx43. The function of gapjunction intercellular communication was tested by Fluorescene recovery afterphotobleatching.3. The hUCBDSCs and the lymphocyte isolated from unrelated donors werecocultured in the presence of PHA-P or not. After48hours, the nonadherent cells wereharvested, washed extensively.The proliferation，apoptosis and cell cycle of lymphocyteswere tested by cell counting kit-8and flow cytometry (FCM).4.We examined the effect of cytokines secretion by co-culturing hUCBDSCstransfected with Ad-Cx43-GFP or Ad-GFP with CD4+T, CD8+T, and human peripheralblood lymphocytes（hUCBDSCs to CD4+T/CD8+T/lymphocytes ratio,1:4）.After cocultedfor72hours, interleukin-4(IL-4) and interferon-γ(IFN-γ) in the culture supernatant werethen analyzed with enzyme linked immunosorbent assay (ELSIA).5.For the regulatory T cells(Tregs), after cocutured72hours, nonadherent cells wereharvested and evaluated for the proportion of Tregs present by flow cytometry usingCD4-PE and CD25-Alex flour647antibodies.Results:1. After transfected by Ad-Cx43-GFP for24h, the expressions of GFP in hUCBDSCswere detected. The rate of transfection was (85±3.2)%after48h. The mRNA and proteinexpressions of Cx43in hUCBDSCs transfected with Ad-Cx43-GFP were higher than thoseuntransfected with Ad-Cx43-GFP (P <0.05). The HUCBDSCs modified by Ad-Cx43-GFP couldpromote the function of GJIC compared with hUCBDSCs transfected by Ad-GFP or not.2. The hUBDSCs promoted or inhibited the proliferation of lymphocytes in a CCK-8assay which depends the ratio of hUCBDSCs to lymphocyte. When the hUCBDSCs tolymphocyte ratio was1:4, the hUCBDSCs could inhibit the proliferation of thelymphocytes. However, when the hUCBDSCs to hPBMCs ratio was1:8, the hUCBDSCscould promote the proliferation of the hPBMCs.3. The hUCBDSCs transfected with Ad-Cx43-GFP caused the increasement of IL-4secretion both in lymphoctes and CD4＋T cells and the decreasement of IFN-γ secretion inlymphocyte and CD8＋T compared to hUCBDSCs transfected with Ad-GFP(P <0.05).4. There was a significant(P <0.05）increase in the CD4+CD25+Tregs population when the lymphocyte were occulted with hUCBDSCs transfected by Ad-Cx43-GFP whencompared with hUCBDSCs transfected by Ad-GFP or not(P <0.05).Conclusions:1.The hUCBDSCs have been shown to participate in key immunological processessuch as the promotion or inhibition of cytokine secretion，the proliferation,apoptosis andcell cycle of the lymphocytes.2. The hUCBDSCs transfected with Ad-Cx43-GFP increased IL-4secretion anddecreased IFN-γ secretion compared with hUCBSDSCs transfected with Ad-GFP, thus weconcluded that the function of hUCBDSCs was improved by enhancing the expressionCx43.3. These data offer insight into the interactions between allogeneic hUCBDSCs andimmune cells and provide theoretical base for the treatment of GVHD.