| Objective:To clone and build the human epidermal growth factor （hEGF） gene eukaryotic expression plasmid in vitro. To Separate the cultured rat adipose stem cells （adipose-derived stem cells, ADSCs）, and observe the transfection efficiency; To establish the rat full-thickness skin lesions model, and transplant the ADSCs joint fibrin glue to the wound, thus to explore theexperimental and theoretical basis of ADSCs on the wound repair of lesions.Method:1.According to the known the EGF sequence from Gene Bank Human cDNA, synthesize EGF gene signal peptide SP （NM001963, nt453-nt518）+EGF gene fragment （NM001963, nt3363-nt3521）. sp-hEGF genes were loaded into puc57carrier, and were detected by sequencing and restriction endonuclease. To digest puc57carrier with Nhel and Sall, whichever is the target gene and had the same restriction enzyme T4DNA ligase expression vector pYr-ads-6to connect the expression plasmid pYr-ads-6-sp-hEGF can transformed into E. coli DH5a, purified plasmid by restriction analysis and sequencing to determine the recombinant plasmid was successfully constructed.2. The separation, culture and identification of ADSCs:SD rats were put to death and the fat pad of groin were obtained, to remove the fascia and blood vessels and cut into pieces with diameter of2mm, the primary cells were gained with collagenase digestion method, then observing cell growth state and morphological changes. After purification and passage, the3rd generation ADSCs which were grew well were collected, then detected CD44expression with immunocytochemical staining to identify the antigen marker.3. To transfect expressed plasmid pYr-ads-6-sp-hEGF into3rd generation ADSCs with lipofectamine method, and observe the proportion of green fluorescent cells under the fluorescence microscope to determine the transfection efficiency.4. The establishment of full-thickness lesions models of rats:30rats were anesthetized by intraperitoneal injection with3%sodium pentobarbital. To produce three1.0cm×1.0cm full-thickness skin lesions wounds on both sides of its spine. The rats were randomly divided into three groups, ADSCs plus fibrin glue group （A）, fibrin glue group （B）, blank control group （C）.40ul cell suspension and fibrin glue were evenly applied to the wounds of the transplant team, the control group was coated with fibrin glue, and no treatment was administered in blank control group. Observe the wound changes on the3rd,7th, and14th days after surgery in each group. The healing tissue was taken, chipped and embedded on the14th day. Observe the morphological changes of each group with HE staining, and changes of expression of CK19and collagen type Ⅲ with immunohistochemistry method.Results:1. The recombinant plasmid pYr-ads-6-sp-hEGF with restriction analysis and sequencing confirmed that the target sequence was inserted correctly, and that containing hEGF gene eukaryotic expression plasmid pYr-ads-6-sp-hEGF was constructed successfully.2. The ADSCs of rats were grown adherently, showing a circle or triangle initially, with many impurities cells.3-5days later, the morphology were like fibroblasts, and proliferated fast in vitro. After passage, the cell morphology was the same with primary state. In the third generation of ADSCs, about87%CD44of cells had positive expression.3.24h after transfection ADSCs observed under a fluorescence microscope to determine the transfection efficiency reached about12%.4.3days after surgery, three groups of lesions had no difference; on the7th day, the wounds of group A were dry, and slightly the other two groups were moist; On the14th day, the wound area of group A was significantly reduced, the surface was smooth; The wounds in Group B scattered only in the skin island formation, with rough wound, Group C had part of the crust bar formation. Histological observation showed group A had thick skin epidermis, the dermis layer of collagen fibers arranged regularly; the epidermis in group B was thin; no epidermis generated in group C. The skin table cortex expression of CK19in Group A increased significantly （P<0.05）, expression of Group B was lower than the transplant group （P<0.05）, Almost no CK19expression in Group C （P<0.05）, There was a significant difference among three groups; type III collagen expression level increased in group A（P<0.05）, no significant difference was showed between group B and group C on type III collagen expression （P>0.05）.Conclusion:1.The rat ADSCs are obtained easily, with enough quantity, and is proliferated stably in vitro.2. The hEGF gene recombinant plasmid is successfully constructed.3.Liposome-mediated method can transfer hEGF gene to ADSCs, but the efficient is lower.4. ADSCs can increase CK19and III collagen expression, thus promote the wound healing. |
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