Preliminary Study On The Mechanism Of ADSCs Promoting The Repairment Of Radiation-induced Dermal Injury By Co-culture Model

Radiotherapy,occupational exposure,accidents,war and other causes can lead to radioactive damage to the skin.Large doses of radiation can cause acute radiation ulcers,and low doses of radiation can cause radiation dermatitis,which showed delayed healing that can cause chronic radiation ulcers.If not timely treatment,and ultimately may lead to radiation skin cancer.The radiation skin lesions are hidden,advancing,and difficult to heal clinically.Radiation skin ulcers may advance into deep tissues,thus damage muscle,blood vessels,nerves etc.Those may affect patients' life quality seriously,and may even turn into life threats.At present,radiation ulcer is the most frequent manifestation of radiation skin injuries clinically.Using flaps to repair skin defects is the most common and effective treatment.In recent years,ADSCs that originate from adipose tissue has been widely used as a research hotspot in regenerative medicine and tissue engineering.ADSCs is a kind of mesenchymal stem cells(MSCs),which have biological characteristics of multiple differentiation potential and immune privilege.Therefore,ADSCs have wide clinical prospects.In many clinical trials and animal experiments,ADSCs are used in the radioactive skin lesions and have promoted tissue repairment significantly.Due to their potential to differentiate into multiple tissues,ADSCs can directly differentiate into tissue cells in the repair process.However,with deeper studies of ADSCs,more and more researchers believe that paracrine plays a leading role in the process of wounds repairment.ADSCs can secrete a large number of cytokines,including growth factors,antiinflammatory factors,etc.They can also release extracellular vesicles(EVs)with specific functions including anti-apoptotic and anti-inflammatory effects,and increase angiogenesis.However,further studies are needed about the mechanism of ADSCs for reversing radiation skin injury,and the safety of clinical use of ADSCs remains controversial.Therefore,we hope to establish the cell model that displays ADSCs repair in the radioactive skin damage.Then ADSCs and radioactive dermal fibroblasts are co-cultured in the special culture dishes and separated by semipermeable membrane.By comparing the results of each culture groups which reflect the influence of ADSCs on repairing radioactive dermal fibroblasts by paracrine,we may learn more about the mechanism that how ADSCs repair radiation-induced dermal injuries.The main studies are as follows:Part I.Isolation and identification of rat adipose derived stem cells and dermal fibroblastsPurpose Separation and extraction of high purity r ADSCs and r Fbs.Method Selected male SD rats each weighed about 200-250 g were given intraperitoneal injection of chloral hydrate and acquired the abdominal subcutaneous adipose tissue that were cut into 2-4mm small pieces and added with mixed enzyme solution.According to the requirements of tissue treatment program,r ADSCs were separated.We got rat abdominal skin tissue,removed subcutaneous mucosa and cut the tissue into 4mm×5mm pieces,which were then put into disperse enzyme solution overnight.The dermis was separated from the epidermis and added with the collagenase solution,through which the r Fbs were isolated.Both of the cells were observed in the inverted microscope and the growth curves were depicted by CCK-8 method.The expressions of CD29,CD44,CD31 and CD45 on the surface of r ADSCs were detected by flow cytometry.The r ADSCs were incubated with different inducible reagents for inducing multi-directional differentiation.r Fbs were identificated by HE staining,vimentin immunohistochemistry identification r Fbs.Results Under the inverted microscope,the morphology of r ADSCs were spindle or polygonal that were similar to fibroblasts and the nucleuses were large and obvious,located in the center with obvious nucleolus.The r Fbs showed long spindle with smaller nucleus in the center and spiral and cauliflower-liked colonies.The results of cell phenotype which were identified by flow cytometry showed that the positive expression rates of CD29 and CD44 were 99.42% and 98.29% respectively,while CD31 and CD45 showed negative results.Inducible culture identified that cells possessed the abilities of osteogenic differentiation and adipogenic differentiation,which confirmed that obtained r ADSCs had a multi-directional differentiation potential.Under the light microscope,HE staining showed that the nucleuses of r Fbs were deep stained and round or oval in the middle with pale staining of cytoplasm.The immunohistochemistry showed that r Fbs had positive vimentin expression and the rate was almost 100%.Both of r ADSCs and r Fbs had strong abilities to proliferate by CCK-8 method.Conclusion The r ADSCs and r Fbs that were extracted by enzyme digestion had high purity and activity,which could be prepared for the cell model.Part II.The effects of radiation on the function of r Fbs in vitroPurpose By establishing radiation injury model of r Fbs,we compared the condition of r Fbs at different doses and times,though which we could determine the reasonable dose and time for the co-culture model.Method The r Fbs were irradiated with doses of 2 Gy,8 Gy,16 Gy,24 Gy,36 Gy by a linear accelerator respectively,and the control group was set without radiation.The growth curve of r Fbs in each group was described by CCK-8 method.The cell cycle and apoptosis of r Fbs were detected by flow cytometry at 2d,4d and 6d after irradiation.Results With the increase of radiation dose,the proliferation ability of r Fbs decreased gradually and the proportion of apoptosis increased.At the same time,the proportion of G1 phase in cell cycle decreased,the ratio of G2 phase increased and the G1 / G2 ratio decreased.In all,the most obvious change was on the second day after irradiation.Conclusion Radiation had many effects on r Fbs including decreased proliferation,increased apoptosis and G2 arrest,in which there was a dose-effect relationship.According to the results,we selected the radiation dose of 8Gy to establish a co-culture model and detected the relevant indicators on the second day after irradiation.Part III.In vivo and vitro test to detect r ADSCs to repair r Fbs after radiation injury by paracrinePurpose The r ADSCs and r Fbs were co-cultured with semipermeable membrane,in which the medium can promote the healing of radioactive skin lesions in rats.The r ADSCs could promote the repairment of r Fbs beside the semipermeable membrane by paracrine,indicating the effectiveness of co-culture model and further study on the repair mechanism of r ADSCs.Method The rat model of radioactive skin injury was established in vivo,Locally injecting r ADSCs,r ADSCs co-culture medium,complete medium around the wound,Observing the change of organizational structure of wound by measuring the area of the wound,HE and Masson staining,PCNA and Tunel used to detect the proliferation and apoptosis of the cells,the expression of CD31 and TNF-? were detected by immunohistochemistry,the level of growth factor VEGF protein was detected by Western blot.In vitro experiments,cell treatment was performed according to the grouping.Set up experimental groups: group A—r ADSCs co-culture with radioactive r Fbs;group B—NRK co-culture with radioactive r Fbs;group C—culture radioactive r Fbs alone;group D—r ADSCs co-culture with r Fbs;group E—culture r Fbs alone.The differences of r Fbs migration ability between groups were compared by scratch experiment.Flow cytometry was used to detect the apoptosis and cell cycle of r Fbs in each group.Ed U was used to detect the cell proliferation ability of r Fbs in each group.Western blot was used to detect the levels of CCND1 and P53 in r Fbs.The levels of TGF-?,GCSF,COL? and COL? in culture medium were detected by ELISA.Results Due to the radiation,the proliferation and migration ability of the r Fbs was harmed with G2 phase arrested,decreased apoptosis,inceased level of P53 protein and TGF-?and decreased level of CCDN1 protein,GCSF,Collagen?and Collagen?.Through the co-culture model,r ADSCs could enhance the proliferation and migration ability of r Fbs significantly,also decreased the apoptosis and the G2 phase arrest with the higher level of CCDN1 protein,GCSF,Collagen?and Collagen?and lower level of TGF-?.Conclusion The co-culture medium of r ADSCs can effectively promote wound healing in rats models.The r ADCSs can improve the proliferation,migration and apoptosis of r Fbs after irradiation by paracrine in co-culture model,through which can repair r Fbs radioactive damage...