Changes Of SSB1 Expression And Its Correlation With NBS1 In The Repair Of Submandibular Gland Radiation Injury In Rats

Objective:To investigate the mRNA and protein expression of single-stranded DNA-binding protein1（SSB1）in the rat submandibular gland tissue and epithelial cells,and to explore the relationship between SSB1 and the repair of DNA double-strand breaks.In addition,to study the relationship and interaction between SSB1 and NBS1 in the repair of DNA double-strand breaks in rats submandibular gland.Methods:1.The model of ⁶⁰Coγradiation injury in SD rats was established.The expression of SSB1 gene mRNA was detected by Quantitative real-time PCR in the submandibular gland of rats in blank control group and at different time points（1hour,2,3,4,5,6,7,8hours）after 12Gy irradiation.The expression of SSB1 gene mRNA and protein in the blank control group and the experimental group that the rats exposure to different doses（3,6,9,12,15Gy）of radiation by qRT-PCR and western blot.2.The rat submandibular gland（SMG）epithelial cells were cultured in vitro.The expression of SSB1 gene mRNA in rat SMG epithelial cells was detected by qRT-PCR after different doses（1,2,4,6,8Gy）of irradiation.Western blot was also used to detect SSB1 and NBS1 protein expression in SMG epithelial cells after irradiation with different doses（1,2,4,6,8Gy）.Each experiment were seted up blank control group.3.The recombinant adenovirus of rat rAdE5-SSB1-1p2shRNA was constructed and further infected into rat SMG epithelial cells（rAd-shRNA group）.In addition,the negative control group（rAd-HK group）and the blank control group（control group）were established.The silencing effect of SSB1 and the expression of NBS1 were detected by Western blot method.4.Rat rAdE5-SSB1-1p2shRNA recombinant adenovirus was further transfected into rat SMG epithelial cells.After silencing the expression of SSB1gene,the cells were divided into rAd-shRNA and rAd-shRNA+IR group after irradiation with different doses（0,6Gy）.In addition,irradiated 6Gy group（IR group）and blank control group（control group）were established.Western blot was used to detect the expression of SSB1 and NBS1 protein.Result:1.After irradiation with ⁶⁰Coγ12Gy,the relative expression of SSB1 mRNA in the submandibular gland of rats reached its peak in 4 hour group,and then decreased gradually（p<0.05）,The difference was statistically significant.After different doses of ⁶⁰Coγirradiation,with the increase of radiation dose,the relative expression of SSB1 mRNAs increased first,and reached the peak value in 9Gy radiation dose group then decreased（p<0.05）.The difference was statistically significant.2.The mRNA expression of SSB1 gene in SMG epithelial cells increased with the increase of irradiation dose,reached the peak at 2Gy group,and then decreased gradually with the increase of irradiation dose.The expression of SSB1and NBS1 protein increased first reached its peak at 2Gy group and then decreased with the increase of irradiation dose.The difference was statistically significant（p<0.05）.3.The relative expression of SSB1,NBS1 protein in rAd-shRNA group was significantly lower than that in rAd-HK and control group（p<0.05）.There was no significant difference between rAd-HK group and control group（p>0.05）.4.After silencing SSB1 gene of SMG epithelial cells,the expression of NBS1 gene protein was down-regulated in rAd-shRNA+IR group and rAd-shRNA group.There were significant difference compared with control group（p<0.05）.The relative expression of SSB1,NBS1 protein in IR group was significantly higher than that in control group（p<0.05）.Conclusion:There is no dose-dependent expression of SSB1 gene mRNA and protein in the repair of radiation injury of rat submandibular gland and SMG epithelial cells.Its expression increased first and then decreased with the increase of radiation dose.SSB1 silencing can downregulate the expression of NBS1,and there is a coordination relationship between the two genes.SSB1may be the key to signal activation of DSB repair pathway in rat submandibular gland.