目的探讨RAD51抑制剂RI-1对MSH2缺陷结直肠癌细胞系的杀伤作用及可能的作用机制。方法 Western blot法筛选MSH2差异表达的结直肠癌细胞系,MTT法检测不同结直肠癌细胞系对RI-1(10、20、30、40或50μmol/L)的敏感性;构建针对MSH2基因的重...目的探讨RAD51抑制剂RI-1对MSH2缺陷结直肠癌细胞系的杀伤作用及可能的作用机制。方法 Western blot法筛选MSH2差异表达的结直肠癌细胞系,MTT法检测不同结直肠癌细胞系对RI-1(10、20、30、40或50μmol/L)的敏感性;构建针对MSH2基因的重组shRNA慢病毒表达载体并感染HT29细胞。RI-1(40μmol/L)处理细胞,流式细胞术检测细胞凋亡的变化;单细胞凝胶电泳实验分析细胞内DNA损伤情况;细胞免疫荧光法检测γ-H2AX foci的形成。结果与对照组相比MSH2缺陷的HCT8细胞有明显细胞凋亡现象(P<0.01);HCT8细胞和HT29 Shmsh2细胞尾部DNA含量、尾长和尾距均较对照组明显增加(P<0.05);HCT8细胞和HT29 Shmsh2细胞内γ-H2AX foci的形成数量较对照组明显增加(P<0.01)。结论 RAD51抑制剂RI-1可能通过增加细胞内DNA损伤参与RI-1对MSH2缺陷肿瘤的杀伤作用。展开更多
The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin ...The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin library was constructed using phagemid expression vector.Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells,and the lipocalin secondary library binding to mast cells was obtained.Then the lipocalin secondary library was enriched and screened with FcεRI-αreceptor protein as target molecule,and specific binding phages were eluted.After three rounds of screening,eight recombinant phage clones were randomly selected from elution clones of the third round.ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-αreceptor of mast cells.These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-αreceptor,and also lay the foundation for the development of biological small molecule drugs to treat Ig E associated allergic diseases.展开更多
基金The Science and Technology Project Foundation of Guangzhou,China(Grant No.201604020106)Natural Science Foundation of Guangdong Province(Grant No.1814050002837)
文摘The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin library was constructed using phagemid expression vector.Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells,and the lipocalin secondary library binding to mast cells was obtained.Then the lipocalin secondary library was enriched and screened with FcεRI-αreceptor protein as target molecule,and specific binding phages were eluted.After three rounds of screening,eight recombinant phage clones were randomly selected from elution clones of the third round.ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-αreceptor of mast cells.These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-αreceptor,and also lay the foundation for the development of biological small molecule drugs to treat Ig E associated allergic diseases.
