The Mechanism Of Connexin26 In Regulating Radiation-induced Changes Of Skin Immunochemokine CCL27

At present,radiotherapy is one of the important means for the treatment of cancer,in which X-rays as low LET rays are one of the critical method for radiotherapy.Heavy ions as high LET rays have not been widely used.Heavy ions are hindered as the main means of radiotherapy because their equipment occupies too much space.Although heavy ions have achieved satisfactory clinical results in head and neck tumors,liver cancer,prostate cancer and other cancers,there are also many disadvantages.Among which acuteradiodermatitis（ARD）,as a frequent complication in the process of radiotherapy,has become one of the key factors hindering radiotherapy.In previous studies,it has been found that the process of skin inflammation is accompanied by the rise of chemokine,which promotes local inflammation through chemokine-mediated immune regulation in vivo.At the same time,our previous studies found that Cx26,as a gap junction protein,plays an important role in the transmission of chemokine CCL27related to radiation-induced skin injury.Radiation is one of the effective methods for the treatment of cancer.At present,X-ray as the principal mode of radiotherapy as low LET rays,the process of producing ARD is not very clear.At the same time,as heavy ions with high LET rays,it is particularly important to explore the occurrence process and mechanism of ARD caused by heavy ions.Part I:Radiation-induced skin damage caused by X-raysObjective:To screen the differential protein expressions in the pathogenesis of acute radiation dermatosis（acute radiodermatitis,ARD）after X-ray irradiation and to explore the pathogenesis of ARD and the possible influence of Cx26.Methods:HaCaT^Cx26-/-cells with knockout Cx26 expression were constructed with CRISPR/Cas9 plasmid.The differential proteins expressions of HaCaT cells irradiated by 0Gy and 5Gy X-rays were screened by protein chip technique.Western blot was used to detect the expressions of inflammation-related MAPK/NF-κB signal pathway and scorch death-related proteins.The occurrence of cell death was detected by flow cytometry.The mouse models of cumulative X-ray irradiation and acute irradiation were established,and the degree of inflammation and injury were observed by HE staining.At the same time,the expression of MAPK signal pathway related protein and NF-κB signal pathway protein in the irradiated group were detected by IF.Results:1.In the irradiation group（5Gy）and the control group（0Gy）,after the original data were normalized by software,the differential protein expressions were screened by foldchange（expression difference multiple）.Foldchange≦0.83 or foldchange≧1.2,and the average（fluorescence）signal value of each group was more than 150.A total of 30 differential proteins were screened,of which the expression of 6proteins increased and 24 proteins decreased.According to GO enrichment analysis,there were 23 differential proteins in cellular composition analysis,1446 differential proteins in biological process classification and 122 differential proteins in molecular functional classification.The selection standard was the number of differently expressed genes located on a term/GO.122 signal pathways related to differential genes were obtained by kegg enrichment analysis.The standard for selection was the case that the number of differential genes on a term/pathway≧5,p-value<0.05.The term/pathway obtained in the drawing was arranged in descending order according to the value of Count,and the first 12 results were taken.2.Compared with HaCaT and HaCaT^Cx26-/-in the irradiation group（5Gy）,the differential protein expressions were screened by foldchange（expression difference multiple）after the original data were normalized by software.Foldchange≦0.83 or foldchange≧1.2,and the average（fluorescence）signal value of each group was more than 150.a total of 24 differential proteins were screened,of which 7 proteins were up-regulated and 17 proteins were down-regulated.According to GO enrichment analysis,there are 28 differential proteins in cell composition analysis,1310 differential proteins in biological process classification and 81 differential proteins in molecular functional classification.The selection standard is the number of genes that differ on a certain term/GO.115 signal pathways related to differential genes were obtained by kegg enrichment analysis.The selection standard was that the number of different genes on a term/pathway≧5,p-value<0.05.The term/pathway in the drawing was arranged in descending order from the size according to the value of Count,and the first 12results were taken.3.Western blot assay showed that the expression of p-p65,p-MEK1/2,p ERK,p-p90RSK and p-JNK in HaCaT^Cx26-/-cells increased significantly with the increase of radiation dose,and the expression of these six proteins in HaCaT^Cx26-/-cells was significantly lower than that in HaCaT cells with normal JNK expression（single sample ANOVA）.4.In the animal experiment,the skin of mice showed different degrees of damage after cumulative irradiation and acute irradiation.HE staining showed that the skin of cumulative irradiated mice thickened,and the skin of acute irradiated mice became thinner than that of unirradiated mice.The two irradiation methods caused the disorder of subcutaneous structure and infiltration of inflammatory cells in subcutaneous,hair follicles,hair stem and so on.In IF test,the fluorescence intensity of p65 and p-ERK in the skin of mice treated with cumulative and acute irradiation increased significantly.Conclusion:1.After X-ray irradiation,the differential proteins of HaCaT cells are enriched in inflammatory response and oxidative stress,suggesting that radiation damage caused by ionizing radiation may play a role through the inflammatory response-related pathway.2.HaCaT cells produced highly pro-inflammatory scorch death response after X-ray irradiation,and the inflammatory signal pathway activated by X-ray stimulation may promote cell death through the pyrogenesis reaction.3.The inflammatory response-related protein of HaCaT^Cx26-/-cells decreased significantly,while the scorch-related protein decreased significantly.Cx26 may be involved in the process of cell death through the inflammation-related signal pathway,and CCL27,as a chemokine downstream of the inflammatory pathway,may be regulated by Cx26.PartⅡ:The effect of heavy ion irradiation on CCL27 of HaCaT cells.Objective:To explore the difference between heavy ion irradiation and X-ray damage and CCL27 secretion in human keratinocytes,to explore the difference between high let radiation and low let radiation on the occurrence of ARD,and to find a way to alleviate ARD caused by heavy ion irradiation.Methods:HaCaT cells were irradiated by ¹²C ions.The amount of CCL27 secreted by the cells was detected by ELISA.Clone survival test to detect the degree of radiation damage of HaCaT,HaCaT,Vector and HaCaT^Cx26-/-cells irradiated by ¹²C ions.Western-blot was used to detect the expression of proteins related to NFκB/MAPK signal pathway after different doses of irradiation.Results:1.¹²C ions caused obvious damaging effect on HaCaT cells,and the secretion of CCL27 is less than that of X-rays.2.Heavy ion irradiation had obvious damaging effect on HaCaT cells,and the ability of cell proliferation and survival decreased with the increase in dose.HaCaT^Cx26-/-cells have higher survival ability and lower radiosensitivity than normal HaCaT cells.Conclusion 1.¹²C ions may produce less inflammatory responses than X rays.2.¹²C ions have obvious radiation damage to HaCaT cells.3.Cx26 plays an important role in the production of ¹²C ions radiation damage signal.