| Objective Burns are common and severe traumata in peacetime and wartime. Thetreatment effect on a batch of burned patients is closely related to the whole socialstability. The key step for rescuing severe burn patients is to cover the wounds as earlyas possible. In recent years, various studies have indicated that mesenchymal stem cells(MSCs) promote the functional recovery of injured tissues and organs. Considering thetherapeutic efficacy of MSCs for the injured tissues and organs, there is great interest inthe potential use of MSCs for treating severe burns. Therefore, the aim of the presentstudy is to evaluate the effect of human umbilical cord mesenchymal stem cells(hUCMSCs) on wound healing of severe burn rats and its potential mechanisms.Moreover, these data might provide the theoretical foundation for the further clinicalapplication of hUCMSCs in burn areas.Methods (1) The hUCMSCs were extracted by three enzymatic methods (collagenaseⅡ, collagenaseⅡ/trypsin and collagenaseⅡ/hyaluronidase) and an explant culture. Theexpression of cell surface markers, such as CD105, CD90, CD44, CD29, humanleukocyte antigen-Ⅰ(HLA-Ⅰ), CD45, human leukocyte antigen class Ⅱmolecules(HLA-DR), CD31and vWF were detected by immunofluorescent staining/flowcytometry. The differentiation potential of these cells was evaluated by the oil red Ostaining, the new blue staining and the Von Kossa calcium deposition methods. The cellmorphology and ultrastructure were observed using the inverted microscope andtransmission electron microscope (TEM). The proliferation of hUCMSCs in differentgeneration was evaluated by3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT). The cell cycles of hUCMSCs in different generation were evaluated byPI staining and flow cytometry.(2) The serum from severely burned patients (BPS) andhealthy controls (HCS) was collected. The hUCMSCs were randomly divided into threegroups:10%FBS,10%HCS and10%BPS at3d. The cells apoptosis was detected byAO-EB staining and flow cytometry. The cell morphology and density were observedby inverted microscope. The cell proliferation activity was assessed by MTT. The cell cycle were evaluated by PI staining and flow cytometry. The cell senescence wasdetected by β-gal staining.(3) The126adult male Wistar rats were randomly dividedinto3groups: sham, burn, and burn transplanted hUCMSCs. The GFP-labeledhUCMSCs/PBS were transplanted through tail vein injection into rats of thecorresponding groups. The rate of wound closure was evaluated by Image Pro Plus.GFP-labeled hUCMSCs were tracked by in vivo bioluminescence imaging (BLI), andhuman-specific DNA expression in wounds was detected by PCR. Inflammatory cells,neutrophils, macrophages, capillaries and collagen types I/III in wounds were evaluatedby histochemical staining. Wound blood flow was evaluated by laser Doppler bloodflow meter. The levels of pro-inflammatory and anti-inflammatory factors, VEGF,collagen types I/III in wounds were detected using an ELISA assay.(4) The hUCMSCswere treated with10%HCS,10%BPS at3d and inhibitor of Notch signal DAPT/GSIor with10%HCS,10%BPS at3d,10%HCS+20ng/mL VEGF,10%HCS+20ng/mLbFGF and10%HCS+20ng/mL VEGF+20ng/mL bFGF. Proliferation numbers ofhUCMSCs were evaluated by MTT and trypan blue exclusion dye. The cells cycle weredetected by PI staining and flow cytometry. The expression of cyclin D was evaluatedby Western Blot. The levels of bFGF, VEGF and other cytokines in the serum weredetected by ELISA. The expression of VEGFR1and bFGFR2was assayed byimmunofluorescence staining. The Notch-1and Hes-1, the key factors of the Notchsignaling were detected by Western Blot and qRT-PCR.Results (1) The hUCMSCs were successfully isolated by four methods. Compared withthe other methods, more cell quantities were obtained by collagenaseⅡ/hyaluronidasemethod. Therefore, the collagenaseⅡ/hyaluronidase was optimal for the isolation ofhUCMSCs. The cell surface markers of interstitial system were expressed, while therewere no cell surface markers of endothelial and hematopoietic systems. The cellmorphology was long spindle and nucleus was large and irregular. These cells can beinduced to be adipocyte, chondroblast and osteoblast through using specific inducingliquid. The result of the cell cycle study indicated that more than80%of the cells werein the stationary phase(G0～G1phase). Based on these above results, the isolated cellswere hUCMSCs. Moreover, The proliferation of hUCMSCs displayed an initial lagphase at1d, an exponential log phase from2d to6d, and a last plateau phase at7d. ThehUCMSCs could inhibit significantly allogenetic T cells proliferation that was inducedby plant haemagglutinin (PHA).(2) The hUCMSCs in10%FBS,10%HCS and10% BPS at3d showed normal morphology and structure, and no apoptotic cells or deathcells were observed. Meanwhile, the results of flow cytometry were similar to theresults. At2d-6d, velocity and numbers of hUCMSCs proliferation in10%BPS at3dwere significantly faster and higher than that in other two groups. Meanwhile, theproliferation results of cells in10%BPS at3d were similar to the MTT results. Therates of proliferation period (S) of hUCMSCs in10%BPS at3d were significantlyhigher than that in other two groups, while the senescent rates of hUCMSCs weremarkedly lower. The higher levels of cytokines in serum from the severely burnedpatients can significantly stimulate hUCMSCs proliferation, prevent cells fromapoptosis and reduce cells senescence. Therefore, it is feasible to use hUCMSCstransplantation for treating severe burn animal model.(3)The hUCMSCs transplantationaccelerated the wound closure of severe burn rats. These cells migrated into wound andremarkably decreased the quantity of infiltrated inflammatory cells and the levels ofIL-1, IL-6, TNF-α and increased levels of IL-10and TSG-6in wounds. Additionally,the neovascularization and VEGF levels of wounds in the hUCMSCs transplanted groupwere markedly higher than those in other control groups. The ratio of collagen types Iand III in the hUCMSCs transplanted group were markedly higher than that in the burngroup.(4) Compared with10%HCS, treatment with10%BPS at3d markedly increasedthe number of hUCMSCs and significantly induced cell cycle progression into the Mphase. And the expression level of Cyclin D was higher than that in the control group.Moreover, Notch-1and Hes-1were overexpressed after esposure to10%BPS at3d.Nevertheless, proliferation numbers of hUCMSCs, rate of proliferaion period (G2/M+S),the expression levels of Cycling D, Notch-1and Hes-1were markedly decreased byNotch signaling inhibitors (DAPT/GSI). The levels of bFGF and VEGF in BPS weremarkedly higher than the other cytokines. Meanwhile, the receptors of VEGF and bFGFwere expressed on the cell surface of hUCMSCs, respectively. Inhibition of bFGF andVEGF in10%BPS at3d using neutralizing antibodies of bFGF and VEGF individuallyor in combination significantly decreased hUCMSCs proliferation. On the contrary, theproliferation of hUCMSCs was induced by the soluble bFGF, VEGF, and a cocktail ofbFGF plus VEGF. The overexpression of both Notch-1and Hes-1at the protein andmRNA levels were also induced. Therefore, bFGF and VEGF in the BPS were keyfactors to promote proliferation of hUCMSCs.Conclusion The hUCMSCs transplantation remarkably accelerated the wound closure of severe burns rats. In addition, bFGF/VEGF in BPS can significantly stimulatesurvival and proliferation of hUCMSCs through activating Notch signaling. |
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