| ObjectiveThis study aimed to investigate the feasibility of using keratinocytes combined with microskin autografting techniques to treat full-thickness skin defect wounds, which would provide a novel technique for clinical study.Methods1. The isolation, cultivation and gene modification of epidermal cellsThe ECs harvested from human epidermal tissue by enzyme digestion method were cultured in vitro.(1) To investigate the best MOI:The cells were transfected with Ad-hEGF in different MOIs (0,5,20,50,100,150 and 200). After 24,48 and 72h transfection, the condition of the cells and the green fluorescent protein expressed by the cells were observed, the transfection efficiency of the green fluorescent protein was detected by flow cytometer, the expression of EGF was detected by ELISA, the growing activity of the cells were detected by CCK8, and to choose the best MOI.(2) To investigate the biological activity of EGF secreted by the transfection cells:The cells were randomly divided into two groups, one was untransfection group, the other was transfection group. The untransfection group was cultured with the normal ECs'liquid supernatant. The transfection group was cultured with the transfection ECs'liquid supernatant (ECs were transfected with Ad-hEGF in the best MOI 50). After 1,3 and 5d cultured, the biological activity of them secreted EGF were measured by CCK8.(3) To investigate the differentiation markers expressed by the transfection cells:The cells were randomly divided into two groups, one was untransfection group, the other was transfection group (the transfection group were transfected with Ad-hEGF in the best MOI=50). The related markers CK14 and CK19 about ECs were measured by immunofluorescence staining at 24h.(4) To investigate the transfection cells' migration:The cells were randomly divided into two groups, one was untransfection group, the other was transfection group (the transfection group were transfected with Ad-hEGF in the best MOI=50). After 0,24 and 48h scratched, the cells'migration was observed and the distance about cells'migration was measured.2. An experimental study on the repair of full-thickness skin defect wounds with graft of keratinocytes and autologous microskinSeventy adult Sprague-Dawley (SD) rats (weight:210-230 g) were randomly divided into seven groups according to the random number table method. There were ten rats in each group, i.e. autologous microskin at an area expansion ratio of 10:1 (group ?), autologous microskin at an area expansion ratio of 20:1 (group ?), autologous microskin at an area expansion ratio of 20:1 in combination with keratinocytes (group ?), autologous microskin at an area expansion ratio of 20:1 in combination with the EGF gene modified keratinocytes (group ?), keratinocytes suspension only (group V), EGF gene modified keratinocytes suspension only (group ?) and control group (group ?). Making a model of anti-contracture of skin defect wound in rats and transplanting correspondent graft into the wound. The wound was covered by the allogeneic skin (allogeneic skin derived from Wistar rats).(1) Gross observation and healing rate of the wound:Observing the survival of rats after operation. Wound repair was observed by gross observation and wound healing rate.(2) The histological examination of the wound:Wound repair was observed by tissue biopsy and immunohistochemistry staining at 21 d post-wounding.(3) The detection of mRNA EGF level in the wound tissue:EGF mRNA expression in wound was measured by Real-time PCR at 7 d,14 d,21 d post-wounding.Results1. The isolation, cultivation and gene modification of epidermal cells(1) To investigate the best MOI:After the transfection of 24h and 48h, the ECs grew well in all groups, like small polygon, the cytoplasm was translucent, the nucleus was larger and in center of the cell. At 72h, the cell debris increased significantly in group MOI 200. The green fluorescent protein was expressed in each transfection group except the untransfection group. At 72h, the transfection efficiency was more than 90% in groups MOIs 50,100,150,200. The transfection efficiency of the untransfection group and groups MOls 5,20 were (0.51±0.20)%, (62.44±6.23)%, (75.00±5.43)%, which were obviously lower than that of group MOI 50[(93.12±2.55)%, with P values of paired comparison below 0.01]. After transfection, the expression of EGF increased gradually. At 72h, the expression of EGF in group MOI=50 was obviously higher than that of other groups (with P values of paired comparison below 0.01). There was no difference in each group about the OD of growing activity at 24,48h (P>0.05). At 72h, the OD of growing activity in group MOI 200 was obviously lower than that of other groups (P<0.05). At Id, there was no difference in two groups about the biological activity of EGF (P>0.05).(2) To investigate the biological activity of EGF secreted by the transfection cells:At 3d and 5d, the transfection group about the biological activity of EGF were 1.10±0.11 and 1.87±0.21, which were obviously higher than that of untransfection group (with P values of paired comparison below 0.01).(3) To investigate the differentiation markers expressed by the transfection cells:At 24h, CK.14 and CK.19 as keratinocyte differentiation markers were elevated in the transfection group, which was higher than that of untransfection group.(4) To investigate the transfection cells'migration:At 48h, the transfection cells had been basically covered the mark, the untransfection cells had not been reached the center of the mark. After 24 and 48h scratched, the migration distance of transfection cells was obviously higher than that of the untransfection group (with P values of paired comparison below 0.01).2. An experimental study on the repair of full-thickness skin defect wounds with graft of keratinocytes and autologous microskin(1) Gross observation and healing rate of the wound:All the rats survived until the experiment was completed. The allograft skins in all groups were survived, dried and removed off. The groups of ?, ?, ?, ?, ? had tracts of visible epithelium. The group of ? had a few thin epithelium. There was no epithelization in group ?. The wound healing rate was obvious lower in group ? (66.0%±6.0%) than that in groups ? (83.2%±5.6%), ? (73.6%±5.3%) and IV (83.7%t4.3%) (with P values of paired comparison below 0.01). There was no difference in group ? and ? about wound healing rate (P>0.05). There was no difference in group ? and ? (66.5%±5.1%) about wound healing rate (P>0.05). The wound healing rate was obvious lower in group ? than that in group ? and IV(with P values of paired comparison below 0.01). The wound healing rate was remarkably lower in group ? (36.5%±6.1%) than that in other groups except group ? (22.7%±5.5%) (P<0.01).(2) The histological examination of the wound:The results of pathology showed that squamous epithelial cells were observed in group ?, ?, ?, ?. The neo-epithelium was thicker and included a lot of vacuoles in group ? and ?. There was no epithelial in group ?. The IHC staining showed that the expression of collagen IV in group ?, ?, ?, ?, ? and ?. Laminin was present in group ?, ?, ? and ?. Collagen ? and laminin were not present in group ?.(3) The detection of mRNA EGF level in the wound tissue:An extremely high EGF mRNA level was observed in group ? and ? at 7 d,14 d compared with other treatment groups. However, the EGF mRNA was almost undetectable in all groups at 21 d post-wounding.Conclusions 1. The suitable MOI were 50-150, and MOI 50 was the best value. The hECs transfected with Ad-hEGF could effectively express the EGF gene highly and keep the good biological characteristics.2. The keratinocytes combined with autologous microskin could increase the wound healing rate. The keratinocytes could elevate the efficiency of large wound healing with treatment of autologous microskin in order to provide a good way which could promote wound healing by using only a tiny of autologous skin.3. EGF highly expressed keratinocytes combined with autologous microskin could improve the wound healing rate greatly. The keratinocytes could effectively secrete EGF to accelerate wound repair and reduce the amount of autologous skin used. However, considering the clinical limitations of wounds treated with EGF gene modified keratinocytes, a novel way to promote EGF highly expressed is needed to develop in future studies. |
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