Study On CCL27 Secretion In The Skin Of Mouse Exposion To Ionizing Radiation By CCR10 Regulation

Objective:Recently,radiotherapeutic technology is developing rapidly,including the innovation of irradiation technology and the change of clinical radiotherapy mode.Although radiotherapy can improve the local control rate of tumor and prolong the total survival time of patients,there are various complications in the process of treatment and post-radiotherapy.Acute radiation skin injury,namely radiation dermatitis,is one common complication,which shows different degrees of damage in 95%patients.Severe radiation dermatitis may lead to interruption of treatment,further influencing the local control rate of tumor.Chemokine?CCL?is one cytokine,belonging to the secretion type of small molecular protein family,which shows chemotaxis for lymphocyte migration and homing to the target site in body.It possesses the capability for immune response and induction of inflammation by combining chemokine receptor.CCR10 is one of chemokine receptors,which produces immune response in skin through combining CCL27.In present study,the C57BL/6 mouse with CCR10 gene half knocked-out(CCR10^+/-)was used as the experimental model.Three kinds of mice with different genes,including CCR10^+/+,CCR10^+/-and CCR10^-/-,were obtained in descendant by CCR10^+/-mice crossing.Furthermore,the levels of CCL27 secretion in the mice with different genotypes were detected after X-rays irradiation in different doses.Methods:The mice with CCR10^+/+,CCR10^+/-and CCR10^-/-genotype were utilized respectively in this study.ELISA method was used to measure the levels of CCL27secretion in the skin of irradiated mice.The alteration of CCL27 secretion was analyzed by the comparison between irradiated mice and control.Irradiation method includes 5Gy single irradiation and 30Gy fractionated irradiation.The concentrations of CCL27 and IL-1?in skin of mice were detected on the same day,3,7 and 10 day after irradiation finished.Meanwhile,some skin samples were collected to observe the distribution of CCL27 in skin and the damage of skin by immunofluorescence staining and HE staining.Additionally,two genotypes of mice were jointed to complete the observation for immune response by bystander effect.This experiment contains three groups of animals,5 mice in each group.After two months,flow cytometry?FCM?was used to assay the balance for immune cells within two jointed mice.One of two jointed mice was irradiated in 5Gy of X-rays after immune cells'balance;another one was protected to radiation by lead shield.ELISA method was used to detect the concentrations of CCL27 and IL-1?in every pair mice.Some skin tissues were utilized to achieve HE staining for observing the inflammation of irradiated animals and non-irradiated animals.Results:Firstly,the results from single irradiation in 5Gy showed that there weren't significant differences among different genotypes mice without irradiation treatment?P>0.05?.Based on single irradiation,the increasing levels of CCL27 in mice skin were found on 3,7 and 10 day after irradiation finished,and the change showed the gradually increasing tendency dependent upon time,which the concen-tration of CCL27 arrived at the top of level?P<0.01?.The concentrations of CCL27 in CCR10^+/-and CCR10^-/-mice skin were higher than that in CCR10^+/+mice skin on 10 day after irradiation finished?P<0.01,P<0.01?.Meanwhile,the result of immune-fluorescence showed that CCL27was mainly distributed in surface of skin,hair shafts and follicles by 5Gy of irradiation.The brighter fluorescence in skin was found on 7 and 10 day after irradiation finished.ELISA test displayed that IL-1?levels in CCR10^+/-?CCR10^-/-mice skin were obviously increased on the same day and 3 day after irradiation finished and recovered normal level on 10 day after irradition finished.The disorder of structure and the infiltration of inflammatory cells were observed by HE staining.Secondly,after 30Gy of fractionated irradiation,the results showed that there were significant reduction of CCL27concentrations in mice on the same day,3 and 7 day after irradiation finished?P<0.01,P<0.01,P<0.01?,and the reduction was gradually recovered to normal level on 10 day after irradiation finished.Moreover,CCR10^+/-?CCR10^-/-mice showed the higher levels of CCL27 in the skin compared with CCR10^+/+mice?P<0.01,P<0.05?.The result from immunofluorescent staining displayed that the weak immunofluorescence of CCL27 was found in the surface of skin on the same day and 3 day after irradiation finished,which the expression of CCL27 was decreased.CCL27 proteins were recruited in the corium layer of skin and hair follicles again,and a few of CCL27 were distributed in the surface layer of skin.IL-1?concentrations were increased in three genotypes of mice on 7 and10 day after irradiation finished.In addition,there were the higher concentrations of IL-1?in irradiated CCR10^+/-and CCR10^-/-mice compared with CCR10^+/+mice on 7 and 10days after irradiation finished?P<0.01,P<0.01?.The result from HE staining revealed that the disorder of skin structure was found in the irradiated CCR10^+/-and CCR10^-/-mice and plenty of inflammatory cells were observed in skin nearby hair shafts and follicles.Thirdly,after general anesthesia,two genotypes of mice,CCR10^+/-CD45.2 and CCR10^-/-CD45.1/2,were jointed by interrupted horizontal mattress suturing skin of back.Two months after surgery,lymphocytes in spleen were collected and measured by FCM.The result showed that the percentages of immune cells in the spleens of CCR10^+/-and CCR10^-/-mice were 99.8%and 99.2%respectively,which indicated the arrival of one balance between two mice.Based on 5 Gy of single irradiation in three pair of jointed mice,including jointed CCR10^+/-and CCR10^+/-?H-H?,jointed CCR10^+/-and CCR10^-/-?H-KO?,jointed CCR10^-/-and CCR10^+/-?KO-H?,the increasing levels of CCL27 and IL-1?were found in the skin of non-irradiated mice compared with those in the same genotype of mice in three pairs of mice unexposure to irradiation,but not in blood?P>0.05?.HE staining result showed that the mild inflammation in skin of non-irradiated mice were found in H-H pair and H-KO pair exposure to irradiation.Conclusion:Firstly,CCL27 secretion in the skin of CCR10 knock-out mouse is increased by 5Gy of single irradiation and ionizing radiation induces inflammation in skin.Secondly,fractionated irradiation results in damage of keratinocytes in skin of mice.Subsequently,the secretion of CCL27 was reduced concomitant with the last increase of IL-1?secretion,followed by aggravation of inflammation.Thirdly,the model for observation of immune response by bystander effect is successfully established.Two months after jointed mice,immune cells can arrive at one balance between two mice by establishment for collateral circulation.There is one significant difference between CCL27 concentration in the non-irradiated mouse of the irradiated pair mice and that of the pair mice unexposure to irradiation.It is indicated that the secretion of CCL27 and inflammation in non-irradiated skin can be also induced by the bystander effect of radiation.