Observation of effect on xenotransplantation using cryopreserved GGTA1/β4GalNT2 double-gene knockout inbred line Wuzhishan miniature pig skin
Objective To observe the effect of xenotransplantation of GGTA1/β4 GalNT2 double-gene knockout inbred line Wuzhishan miniature pig skin preserved in liquid nitrogen.Methods Cryopreserved double-gene knockout pig split skin and commercially gene transfected pig skin were used.The double-gene knockout pig skin was preserved by vitrification method,and the skin was thawed when used for xenotransplantation.The receptor were rhesus monkey,which were divided into experimental group and control group,with 4 animals in each group,and four pieces of 4 cm×4 cm skin were removed on each monkey′s back,forming a full-thickness skin excision model.Cryopreserved double-gene knockout pig skin and gene transfected pigs kin were transplanted respectively.The survival of the skin was observed at different time points after the operation,and the vitality of the skin was evaluated by color,texture,etc.And samples were taken for pathological examination.Serum was taken before operation and on the 4th,7th,10th,14th and 21st days after operation to detect IL-2,IL-4,IFN-γ,and IL-12 content.Results The survival time of pig skin in the experimental group was[10-21(14.5±3.4)d],significantly longer than[7-14(10.6±2.7)d]in the control group(t = 3.272,P = 0.004).The skin grafts of both groups survived well on the 4th day after operation.On the 7th day after operation,there were small blisters on 2 skin grafts in the experimental group,and big blisters on 6 skin grafts in the control group.On the 10th day after operation,5 skin grafts in the experimental group were completely torn off,6 and 4 skin grafts were still warm and soft in the experimental and control group respectively.On the 12th day after operation,3 and 1 skin grafts in the experimental group and the control group were still warm and soft.On the 14th day after operation,one skin graft in the experimental group remained warm and soft,while all skin grafts lost viablity in the control group.Pathological observation showed that the histological morphology of the skin grafts in both groups were normal on the 4th day after operation.On the 7th day after operation,the structure of skin epidermal cells in the experimental group was complete,while the number of epidermal cells in the control group was reduced.In a part of the skin grafts in both groups,engraft could be seen between the skin and wound bed.On the 10th day after operation,the number of epidermal cells in the experimental group decreased,and the interface between epidermis and dermis was partially separated,while the number of epidermal cells in the control group was further reduced compared with before,and the phenomenon of engraft still existed.On the 14th day after operation,the number of epidermal cells was significantly reduced and the phenomenon of engraft still existed in the experimental group.While infiltration of inflammatory cells in the wound bed could be seen in the control group.Serum IL-2 and IL-4 levels on the 10th day after operation,serum IFN-γ before operation and on the 4th day after operation,and the IFN-γ/IL-4 on the 14th day after operation in the experimental group were higher than those in the control group.Conclusion Cryopreserved GGTA1/β4GalNT2 double-gene knockout inbred line Wuzhishan miniature pig skin survives well after transplantation,and can obtain a relatively long survival time.
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