摘要目的 研究沉默大鼠颌下腺(submandibular gland,SMG)细胞单链DNA结合蛋白1(single-strand DNA-binding protein 1,SSB1)表达后,放射损伤下细胞的增殖及修复情况.方法 机械胰酶联合法进行大鼠SMG细胞原代培养及免疫组织化学法鉴定.实验分3组:正常细胞组、阴性对照组(Ad-HK组)和实验组(Ad-SSB1-shRNA组).重组腺病毒介导shRNA转染SMG细胞,荧光定量PCR检测SSB1表达沉默情况.CCK-8法及免疫荧光法分别检测细胞活性及γ-H2AX焦点数动态变化.结果 细胞免疫组织化学检测角蛋白和α-淀粉酶表达均呈阳性.转染72 h后转染效率接近90%,实验组SSB1 mRNA表达较正常细胞组明显降低(t=16.24,P<0.05).照射前,实验组细胞活性下降不明显,120 h时细胞活性才明显低于正常细胞组(t=3.29,P<0.05).5 Gy照射后,实验组各时间点细胞活性均明显低于阴性对照组和正常细胞组(F=10.19~30.13,P<0.05).γ-H2AX免疫荧光显示,沉默SSB1能抑制细胞DNA放射损伤修复.结论 沉默SSB1表达的大鼠SMG细胞对放射损伤更敏感.SSB1在大鼠SMG细胞DNA放射损伤修复中有着重要作用.

abstractsObjective To investigate the effects of silencing the gene of single-stranded DNA-binding protein 1 (SSB1) on proliferation and DNA repair of rat submandibular gland (SMG) cells after irradiation, and explore the relationship between SSB1 and DNA damage repair. Methods Primary rat SMG cells were obtained by mechanical-enzyme digestion and identified by immunohistochemistry. The cells were divided into three groups, including blank control, negative control and shRNA transfection group. The shRNA was transfected into cells by recombinant adenovirus vector. Real-time quantitative PCR ( qRT-PCR) was used to detect the expression of SSB1 after silencing. The cell viability was detected by CCK-8 assay. Immunofluorescence analysis was performed to observe the dynamic formation of γ-H2AX foci. Results The SMG cells were positively stained for both Pan CK and α-Amylase. The efficiency of shRNA transfection was about 90%at 72 h post-transfection. Compared with the blank control group, the expression of SSB1 was significantly decreased in the cells transfected with shRNA (t=16. 24, P<0. 05). The cell viability of shRNA transfection group without irradiation was decreased indistinctively and became lower than the blank control group significantly until 120 h(t=3. 29, P<0. 05). After radiation with 5 Gy of γ-rays, the cell viability of shRNA transfection group was lower than that of the control groups significantly (F=10. 19-30. 13, P<0. 05). Silencing the expression of SSB1 could increase the number ofγ-H2AX foci in SMG cells at different time of radiation. Conclusions After silencing of the expression of SSB1, the SMG cells could be more radiosensitive, which indicats that SSB1 may play an important role in DNA damage repair after radiation.