| BackgroundRadiation skin damage is a frequent complication of nuclear explosions,radiological nuclear accidents,nuclear fuel handling and clinical oncology radiation therapy.Approximately 87% of clinical patients treated with postoperative radiotherapy for breast cancer present with varying degrees of skin toxicity damage.There are various modalities for treating radiation skin damage in China and abroad,among which Adipose Derived Stem Cells(ADSCs)have been shown to promote the healing of radioactive skin ulcer wounds through their paracrine function.However,adipose stem cells are tumorigenic and highly immunogenic,which may limit their clinical use,and researchers have focused on one of their paracrine products,exosomes.Adipose stem cell exosomes have now been shown to have a beneficial effect on wound repair,are non-cellular in structure,have the advantage of low immunogenicity,and contain the genetic material of the parent cell,which could potentially replace stem cells as a new therapeutic tool.In order to investigate the effect of adipose stem cell exosomes on radiation skin ulcers,animal models of radiation skin ulcers are needed.Various current skin ulcer models include the rat buttock and hind limb ulcer model,the mouse hind limb ulcer model,and the pig skin ulcer model.However,rat and mouse skin ulcer models do not guarantee complete animal survival,with a mortality rate of 12-20%.Although pigs can mimic human skin conditions well without animal mortality,they are larger,more difficult to keep,and have fewer detectable antibodies,which are not conducive to follow-up studies.Therefore,there is an urgent need to establish a replicable,stable,low mortality model of radioactive skin ulcers in small animals.PurposesThe aim of this project is to establish a stable and reproducible animal model of radioactive skin ulcers that simulate acute and chronic skin injury patterns in humans.To investigate the therapeutic effects of adipose stem cell exosomes on radioactive skin ulcers and to lay the foundation for further exploration of the repair mechanism of radioactive skin ulcers.Methods1.The mouse irradiated stent was established with the assistance of3 D printing technology,which is made of polylactic acid(PLA)material.The scaffold is divided into two parts,the irradiated area and the non-irradiated area..2.The mice were randomly divided into two groups.The direct irradiation group was irradiated with 5,10 and 15 Gy doses to the back skin.Using the stent group,the mice were brought to the radiation area and the skin of the back was pulled out separately and irradiated with 15,30 and 45 Gy doses.3.After radiation,the survival of mice in both groups was recorded,survival curves were plotted,and body weight changes were measured.Small intestine hematoxylin-eosin staining was performed to assess the effect of radiation on them.4.Routine photographs were taken to assess wound healing,HE to detect dermal condition,and Masson trichrome to detect collagen thickening.5.Adipose tissue from clinical liposuction patients was collected,primary adipose stem cells were cultured,and the 3rd generation cells were taken to detect surface markers by flow cytometry technology,and then adipose stem cells were subjected to lipogenic and osteogenic differentiation experiments and their identification.6.Extraction of adipose stem cell exosomes by ultra-high speed centrifugation.The morphology and diameter size were analyzed by transmission electron microscopy and nanoparticle size,and the expression of surface proteins CD9,CD81 and TSG101 were detected by protein immunoblotting,and the exosome protein concentration was detected by BCA protein kit.7.Skin tissues of fetal rats were taken,fibroblasts were cultured and co-cultured with adipose stem cell exosomes and fibroblasts,and laser confocal microscopy traced the uptake of PHK26-labeled exosomes by fetal rat skin fibroblasts.8.In vivo experiments were used to verify the effect of ADSCs-derived exosomes on post-radiation injury in mouse skin:Continuous photographs were taken to assess wound healing,ultrasound Doppler to detect blood flow,HE staining to assess dermis thickness,and Maason three-color staining to evaluate skin collagen content.Enzyme-linked immunosorbent assay was performed to detect the levels of basic fibroblast growth factor,transforming growth factor-β1,and vascular endothelial cell growth factor.Results1.In this experiment,an animal model of radioactive skin ulcers in mice was successfully developed with the aid of 3D printing technology.2.The irradiation dose of 45 Gy was determined as the optimal irradiation dose for skin ulcers,and all animals developed radioactive skin ulcers,while the mortality rate of mice was 0%.3.In this experiment,adipose stem cell exosomes were successfully extracted by ultra-high speed centrifugation method to meet the standard.4.This experiment successfully extracted fetal mouse primary fibroblasts and verified that adipose stem cell exosomes labeled by PKH26 membranous dye could be phagocytosed by them into the fibroblasts and around the nucleus.5.Being used in vivo experiments,we verified that adipose stem cell exosomes can promote the healing of radioactive skin wounds and improve collagen and dermal thickening.Conclusions:1.In this study,an animal model of acute radioactive skin ulcers with low mortality in mice was successfully established.2.The irradiation dose of 45 Gy was determined for the durable appearance of radioactive skin ulcers on the back of mice.3.For the first time,it was confirmed by animal experiments that adipose stem cell exosomes can promote the healing of radioactive skin ulcer wounds in mice. |
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