脂肪间充质干细胞与甲泼尼龙联合用药对小型猪异体皮肤移植的影响

Effect of Adipose Mesenchymal Stem Cells in Combination with Methylprednisolone on Allogeneic Skin Grafts in Minipigs

旨在探究脂肪间充质干细胞(adipose derived mesenchymal stem cells,ADSCs)与甲泼尼龙(methylprednisolone,MP)联合用药对小型猪异体皮肤移植(allogeneic skin graft,ASG)所产生的免疫排斥反应与创口愈合的影响。对12头小型猪建立同种异体皮肤移植模型,根据术后对异体皮肤移植皮片不同的干预方式,将小型猪随机分为4组:异体皮肤移植组(ASG)、甲泼尼龙组(MP)、脂肪间充质干细胞组(ADSCs)、脂肪间充质干细胞与甲泼尼龙联合用药组(ADSCs+MP)。术前与术后7、14 d采集血液和组织样本,术后即刻及术后7、14、21 d拍照记录移植表皮临床变化情况。通过对移植皮片表观检查、病理组织学检测,血常规以及血清生化,移植皮片组织氧化应激、炎症、再生的指标检测,综合评价4组不同干预方式对小型猪异体皮肤移植所产生的免疫排斥反应与创口愈合方面的影响。从皮片表观与病理学分析可知,ASG组移植皮片存活时间在7 d左右、MP组移植皮片存活时间在7~14 d、ADSCs组与ADSCs+MP组存活时间为14~21 d。并且在7、14 d时ADSCs+MP组的GSH、SOD的表达量显著升高,在免疫排斥方面,ADSCs+MP组的CD4^(+)T细胞的数量,NF-κB的蛋白与基因表达量,IL-2、IFN-γ、TNF-α、IL-1β、IL-6促炎因子的表达量相对于ASG组显著降低。在创口愈合方面,ADSCs+MP组相对于ASG组VEGF、TGF-β的蛋白表达,SDF-1、CXCR4的表达显著上升。并且ADSCs+MP组相对于ASG组,PI3K、AKT、11β-HSD1的蛋白与基因的表达量显著降低。综上所述:ADSCs+MP组相对于ASG组与MP组,可以延长移植皮片存活时间。并且ADSCs+MP可以有效通过促进GSH、SOD的表达抑制氧化应激。在排异方面,ADSCs+MP可以通过抑制NF-κB的表达抑制IL-2、IFN-γ、IL-6、TNF-α等炎性因子的表达,从而抑制移植过后的免疫排斥反应。并且ADSCs+MP可以通过促进VEGF、TGF-β等愈合因子的表达促进创口愈合。进一步研究发现ADSCs可能通过PI3K/AKT来影响11β-HSD1的表达,进而抑制皮肤皮质醇含量,并促进TGF-β、VEGF等生长因子的表达来促进创口肉芽组织再生与血管迁移。

The aim of this experiment was to investigate the effect of the combination of adipose derived mesenchymal stem cells(ADSCs)and methylprednisolone(MP)on immune rejection and wound healing in allogeneic skin grafts from miniature pigs.An allogeneic skin graft model was established for 12 minipigs,and the minipigs were randomly and equally divided into four groups according to different postoperative interventions on allogeneic skin graft skin pieces:allogeneic skin graft group(ASG),methylprednisolone group(MP),adipose mesenchymal stem cells(ADSCs),and combined adipose mesenchymal stem cells and methylprednisolone(ADSCs+MP)group.Blood and tissue samples were collected preoperatively and at 7 and 14 d postoperatively,and clinical changes of the grafted epidermis were photographed and recorded immediately,at 7,14 and 21 d postoperatively.The effects of the four different interventions on the immune rejection and wound healing of allogeneic skin grafts in minipigs were evaluated by the phenological examination,pathological histological examination,blood and serum biochemistry,and the indexes of oxidative stress,inflammation and regeneration of the grafted skin tissues.From the skin slice phenotypic and pathological analysis,it was observed that the survival time of the grafted skin slice was around 7 d in the ASG group,7-14 d in the MP group,and 14-21 d in the ADSCs and ADSCs+MP groups.and the expression of GSH and SOD was significantly higher in the ADSCs+MP group at 7 and 14 d.In terms of immune rejection,the ADSCs+MP group had significantly higher CD4^(+)T cells,protein and gene expression of NF-κB,and expression of IL-2,IFN-γ,TNF-α,IL-1β,and IL-6 pro-inflammatory factors were significantly lower in the ADSCs+MP group relative to the ASG group.In terms of wound healing,the ADSCs+MP group significantly increased the protein expression of VEGF and TGF-β,and the expression of SDF-1 and CXCR4 relative to the ASG group.the ADSCs+MP group could significantly inhibit the protein and gene expression of PI3K,AKT,and 11β-HSD1.(Conclusion)In conclusion,the ADSCs+MP group could prolong the survival time of grafted skin pieces relative to the ASG and MP groups.And ADSCs+MP could effectively inhibit oxidative stress by promoting the expression of GSH and SOD.In terms of rejection,ADSCs+MP can inhibit the expression of inflammatory factors such as IL-2,IFN-γ,IL-6 and TNF-αby suppressing the expression of NF-κB,thus suppressing the immune rejection reaction after transplantation.Moreover,ADSCs+MP could promote wound healing by promoting the expression of healing factors such as VEGF and TGF-β.Further study revealed that ADSCs may affect the expression of 11β-HSD1 by inhibiting PI3K/AKT,which in turn suppresses skin cortisol content and promotes the expression of growth factors such as TGF-βand VEGF to promote wound granulation tissue regeneration and vascular migration.