The Effect Of Porcine Adipose Derived Stem Cells Overexpressing FTO On Skin Wound Healing

Skin wound healing is an important direction for trauma research.Using tissue engineering technology to construct skin substitutes is an important method to promote wound repair.The key is to find the ideal seed cells.Although embryonic stem cells,bone marrow mesenchymal stem cells,etc.have self-renewal and differentiation pluripotency,they are limited in their widespread use due to the small number of cells,difficulty in obtaining them.damage to the donor,and ethics.Adipose-derived stem cells(ADSCs)are ideal tissue-engineering seed cells due to their wide sources,abundant numbers,rapid proliferation,and low immunogenicity.Recent research has shown that genetically modified stem cells can improve stem cell function,thereby enhancing their clinical treatment effect.Therefore,screening suitable genes for stem cell modification can be used as a new strategy for the rapid repair of skin wounds.Previous research by our group found that FTO can significantly promotes cell proliferation and differentiation,but the function of FTO-modified f ADSCs in skin wound repair has not been reported.Therefore,based on our previous work,we investigate the effects of FTO(Fat mass and obesity-associated protein)on the proliferation and differentiation of porcine adipose stem cells(pADSCs),and further constructs skin cut and burn models on porcine to explore the effect of pADSCs overexpressing FTO on skin wound repair.The results are as follows:1.pADSCs overexpression of FTO promotes cell proliferation and differentiationThe adipose tissue of a 3-day-old Duroc × Landrace × Yorkshire piglet was selected to obtain pADSCs by digestion with type Ⅱ collagenase.After culturing at 37℃ for 3 days,the results show that the cell morphology and proliferation rate were normal.After adipogenesis,the mRNA expression levels of the key adipogenesis related genes PPARy,FABP4,and C/EBPa was significantly increased(P<0.05).Oil lipid O staining resluts showed significant lipid droplets,indicating the successful isolation of pADSCs.Overexpression of FTO in pADSCs promoted the expression of cyclin CDK2 and CDK4(P<0.05),and promoted the transition from the G1 phase to the S phase of the cell cycle(P<0.05).Besides.the pADSCs overexpressing FTO increased the lipid droplets,and the key adipogenesis related genes PPARy,FABP4,and C/EBPα(P<0.05).2.pADSCs overexpression of FTO promotes rapid repair of skin cutsA 32-day-old Duroc × Landrace × Yorkshire piglet was selected for the experiment.Eight wounds were prepared for each pig and divided into 4 groups:control group(Ctl),collagen sponge scaffold group(CSS),and pADSCS combined with collagen scaffold group(CSS/pADSCs).And pADSCS overexpressing FTO combined with collagen sponge scaffold group(CSS/OE-FTO-pADSCs),two replicates in each group,a total of 8 pigs were tested.Construction of skin cut model:A 16 mm diameter stainless steel ring drill was used to prepare a full-thickness skin cut model on the back of piglets.The results showed that the full-thickness skin was completely peeled off and the edges were neat,indicating that the model was successfully constructed."PADSCs-FTO-collagen sponge scaffold" tissue engineering skin construction:The collagen sponge scaffold was prepared by the freeze-drying method.The scanning electron microscope characterization results showed that the collagen sponge scaffold was porous.Inoculation of pADSCs overexpressing FTO into the collagen sponge scaffold could attach and Normal proliferation,indicating that the"pADSCs-FTO-collagen sponge scaffold" tissue-engineered skin was successfully constructed.Effect of "pADSCs-FTO-collagen sponge scaffold" on the repair of skin cuts:In terms of the inflammatory response,on the 7th and 14th days,compared with the control group,CSS group,and CSS/pADSCs group,CSS/OE-FTO group show significantly reduced inflammatory cell infiltration at the wound site(P<0.05);mRNA expression levels of pro-inflammatory genes IL-la and IL6 was decreased significantly(P<0.05);in epidermal and dermal repair,on day 21,compared with the control group,CSS group,and CSS/pADSCs group,the thickness of the epidermis at the wound site in the CSS/OE-FTO-pADSCs group was significantly increased(P<0.05),and the dermal papilla layer was completely formed.In terms of angiogenesis,compared with the control group,CSS group,and CSS/ADSCs group,the blood vessel density at the wound site in the CSS OE-FTO-pADSCs group increased significantly(P<0.05).In terms of collagen deposition,on the 21st day,compared with the control group,CSS and pADSCs groups,the collagen deposition in the CSS/OE-FTO-pADSCs group increased significantly(P<0.05).In terms of wound healing rate,on the 21st day,compared with the control group,CSS group,and pADSCs group,the wound healing rate in the CSS/OE-FTO-pADSCs group was significantly increased(P<0.0 5).The above results show that transplantation of the "pADSCs-FTO-collagen sponge scaffold" can promote skin wound repair and accelerate wound healing by increasing 15%wound healing rate.3.pADSCs overexpression of FTO promotes rapid repair of skin burns32-day-old Duroc×Landrace×Yorkshire piglet was selected for the experiment.Six wounds were prepared for each pig and divided into three groups:control group(Ctl),pADSCS compound 3D hydrogel group(pADSCs group),and pADSCS overexpressing FTO and compounding.In the 3D hydrogel group(OE-FTO-pADSCs),two replicates of each group were tested.A total of 8 pigs were tested.Skin burn model construction:YLS-5Q burner was used to prepare the burn model.When the temperature was 140℃,the time was 30 s,and the pressure was 1500 g,the skin structure of the burn site was damaged,the epidermal layer completely fell off,and the thickness of the dermal layer suddenly changed.Reduced to 1/2,it can be judged that the deep Ⅱ° burn model was successfully constructed.”PADSCs-FTO-3D hydrogel" tissue-engineered skin construction:pADSCs that overexpress FTO were inoculated in VitroGelTM 3D-RGD hydrogel,cultured at 37℃ for 4 h to be semi-coagulated,and cultured for 3 days to prepare"pADSC s-FTO-3D Hydrogel "for tissue-engineered skin.Effect of "pADSCs-FTO-3D hydrogel" on the repair of skin burns:In terms of the inflammatory response,on the 28th day.compared with the control and pADSCs group,there was no bleeding at the wound site in the OE-FTO-pADSCs group,and inflammatory cell infiltration were decreased significantly(P<0.05);in terms of epidermal and dermal repair,compared with the control group and pADSCs group,the thickness of the epidermal layer at the wound site in the OE-FTO-pADSCs group was increased significantly(P<0.05).In terms of angiogenesis,compared with the control group and pADSCs group,on the 28th day,a large number of capillaries and mature small and medium blood vessels were formed in the wound site of OE-FTO-pADSCs group,and the blood vessel density was increased significantly(P<0.05)In terms of collagen deposition,compared with the control group and pADSCs group,on the 28th day,the collagen deposition amount at the wound site in the OE-FTO-pADSCs group was increased significantly(P<0.05).In terms of wound healing rate,compared with the control group and the pADSCs group,the wounds in the OE-FTO-pADSCs group were significantly reduced(P<0.05).The above results show that transplantation of "pADSCs-FTO-3D hydrogel" can promote skin trauma repair and accelerate wound healing by increasing 20%wound healing rate.In summary,pADSCs overexpressing FTO can significantly reduce the inflammatory response and promote epidermal and dermal repair,vascular regeneration,and collagen deposition.