Study On The Repair Mechanisms Of Adipose Tissue Derived Stem Cells In Radiation Injury Of Fibroblasts

The risk of human exposure to radioactive rays is greatly enhanced by nuclear leakage, radiation damage caused by radiation therapy and radiation sterilization in the manufacturing industry. Radioactive skin injury is the most common complication on radiation therapy, it refers to the radiation-induced skin area visible lesions which caused by many kinds of radioactive elements. Common radioactive elements including X-ray,gamma,proton, particle, electronics and other types of ionizing radiation. Because of the hight incidence of radiation-induced skin injury, if you do not take protective measures,about 90% of patients on radiation therapy will appear skin erythema, severe skin loss occurs, ulcers, which affecting the quality of life. The main cells in dermis and connective tissue are composed of fibroblasts （Fibroblasts, Fb）, which play a leading role in wound repair.Adipose-derived stem cells （ADSCs） have multiple differentiation potential, and it is convenient obtainment and can secrete a variety of cytokines to repair the damage. It may have close connection between ADSCs and the repair of radiation damage,It can secrete a variety of cytokines to repair radiation damage or induce paracrine effection of other cells. It is not yet clear that the way in which the cytokines participate.It is known that phosphatidylinositol 3-kinase-protein kinase B （PI3K-Akt） andmitogen activated protein kinase （MAPK） pathway play an important role in many biological research. In recent years, long non-encoding RNA （LncRNA） also play an important role in the field of radiation, the interaction between some LncRNA and PI3K-Akt pathways is also be of concem.It is not clear.Whether LncRNA is involved in the repair mechanism of radiation injury.Based on the above considerations, this study intends to study the repair mechanism of ADSCs in radiation damage by high-throughput sequencing and gene silencing.Part 1 primary culture of ADSCs and Fb , Establishment of co-culture model and the effection of ADSCs on Fb after radiation injuryObjective: To study the effection of Fb on proliferation, apoptosis and cell cycle after radiation intervention through establish the co-culture model of ADSCs and FbMethods: ADSCs and Fb were extracted from rat inguinal fat and dermis by enzymatic digestion method and identified by flow cytometry, multi-directional differentiation and immunocytochemistry. Use 0.4μm Transwell co-culture plate to establish the non-contact co-culture model, We divided into 4 groups, Fb1 group refers to fibroblasts in control group, F2 group refers to fibroblasts with 8Gy x-ray irradiation, Fb3 group refers to fibroblasts recepted by the intervention of ADSCs. NRK group refers to fibroblasts recepted by the intervention of NRK cells. Cell proliferation was detected by CCK-8 assay, apoptosis and cell cycle was detected by flow cytometry.Results: The enzyme digestion method can extract high purity of primary cells, and it identified as ADSCs whose surface marker （CD29, CD44, CD31 and CD45） meet the standards and the extracted ADSCs can be induced into osteoblasts and adipocytes. The high expression of vimentin was detected in extracted fibroblast cells. Fibroblast proliferation in Fb2 group after radiation injury was lower than that of Fbl control group.significantly, the difference was statistically significant （P< 0.05）. The proliferation of firoblasts in Fb3 group was was significantly increased compared with Fb2 group through the intervention of ADSCs, and the difference was statistically significant （P< 0.05）. The apoptosis rate of Fb2 group was significantly increased compared with Fbl group, the difference was statistically significant （P< 0.05）, the ADSCs group co cultured with Fb3,the apoptosis of fibroblasts was significantly lower than Fb2 group, and the difference was statistically significant （P< 0.05）. The cell cycle of fibroblasts was blocked in the G2 phase after 8 Gy irradiation, and the cell cycle in Fb3 group was significantly faster than the Fb2 group after ADSCs intervention, and the cell cycle progressed from G2/M to G1/S. the difference was statistically significant （P< 0.05）.Conclusion: after 8Gy X irradiation, the proliferation activity of fibroblasts was decreased, the apoptosis was increased, and the period was blocked in G2 phase. When we use co-culture model with ADSCs after radiation, the proliferation activity of fibroblasts was increased, apoptosis was decreased, and the process of cell cycle was increased.Part2 The effect on radiation-induced skin injury in rats with the intervention of ADSCsObjective: To study the survival of ADSCs in radiation- induced rats skin injury model and the apoptosis induced by radiation-induced skin injury in rats.Methods: we established the modified Rifkin LH skin radiation model and divided into 5 groups: blank control group, irradiation group, ADSCs intravenous injection group,ADSCs injection group and PBS local injection groupIrradiation area（2cm×2cm） was exposed to 20Gy radiation,ADSCs injection quantity is 2×106, and Luciferase and GFP double labeled lentiviral was transfection. The survival of ADSCs in vivo was observed by using small animal imaging technique at different time points, and frozen sections were observed under fluorescence microscope. Paraffin sections were stained with Tunnel.Results: in the ADSCs group, the lung was intercepted, and the 24h imaging showed that the fluorescence disappeared. The ADSCs local injection group was still able to detect the fluorescence at least 15 days after the injection. Compared with the blank control group, the apoptosis rate of the irradiation group was significantly increased, and the difference was statistically significant （P < 0.05）. ADSCs local injection group was significantly lower than that of simple irradiation group, the difference was statistically significant （P < 0.05）. The apoptosis rate of ADSCs group was lower than that of simple irradiation group （P < 0.05）, the difference was statistically significant.Conclusion: local jection of 2 ×106 , ADSCs can survive for at least 15 days after 20Gy irradiation in rats,and ADSCs can reduce apoptosis induced by radiation-induced skin injury.Part3 High throughput mRNA/LncRNA sequencing, protein mass spectrometric detection and validation with the intervention of ADSCs for Fb radiation injuryObjective: To screen the differential expression of gene and protein levels before and after radiation injury in Fb with the co-culture intervention of ADSCs, and predict the possible signaling pathway.Methods: the experiment was divided into blank group （Fb1 group） , 8Gy group（Fb2 group） and intervention group by ADSCs （Fb3 group） . High throughput mRNA/LncRNA sequencing and protein mass spectrometry were used to screen differentially expressed genes and proteins, and to analyze the possible signaling pathways by sequencing and protein mass spectrometry. Finaly, we screen LncRNA and predict target genes.Results: compared with the blank control group, the P53 pathway in Fb2 group was significantly increased after 8Gy irradiation. The PI3K-Akt and MAPK pathway in the Fb3 group were significantly higher than the 8Gy group treated with the intervention of ADSCs. Real time-PCR and Western-blot were used to verify the related pathway,apoptosis and cell cycle molecules. Fb1 P53,P21,P27,CyclinB, Bax and Cleaved Caspase-3 were increased in Fb2 vs Fb1. The difference was statistically significant （P <0.05）, Cyclin D1 and Bcl-2 were decreased in Fb2 vs Fb1, and the difference was statistically significant （P < 0.05） . Cyclin D1, Bcl-2 and c-myc were increased in Fb3 vs Fb2, and the difference was statistically significant （P < 0.05）. CyclinB, Bax, Cleaved Caspase-3, P21 and P27 were decreased in Fb3 vs Fb2, and the difference was statistically significant （P<0.05）. From the screening of 40 LncRNA combined with Real time-PCR verification, we verify the most four significant changes（LNC₀00473,ENSRNOT00000076586, LNC₀01034 and LNC₀01030）.Conclusion: fibroblasts may be mediated by P53 pathway after 8Gy irradiation.ADSCs may be repaired by PI3K-Akt and MAPK pathway activation. LNC₀00473,ENSRNOT00000076586, LNC₀01034 and LNC₀01030 may play an important role in the repair of radiation injury through the intervention of ADSCs.for Fb.Part4 Study on the mechanism of the repair through the intervention of ADSCsObjective: To detect the differential cytokines secreted by co-culture model, and to investigate the effects of cytokines on the proliferation, apoptosis and cell cycle changes after radiation injury. Preliminary study on the role of LncRNA₄73 in the repair through the intervention of ADSCs .Methods: the expression of cell factor and Elisa to verify the differences in the supernatant of cell culture factor protein chip screening. The proliferation, apoptosis and cell cycle of fibroblasts were detected by the screening of cytokines, We reveal the cytokines activation in signal pathway. Fibroblasts were transfected with shRNA to silence LncRNA₄73, and the proliferation and apoptosis of fibroblasts were detected after A Co-culture.of ADSCs.Results: the expression of p-Akt and p-Erk in GM-CSF group and VEGF group was significantly higher than that in control group （P < 0.05）, which might play a role in the activation of PI3K-Akt and MAPK pathway. at the same time, the proliferation of fibroblasts was significantly lower than that of fibroblasts with the intervention of ADSCs.the difference was statistically significant （P < 0.05）.Conclusion: GM-CSF, VEGF and LncRNA₄73 may play an important role in the repair of radiation injury with the intervention of ADSCs.