摘要
目的:构建表达突变型人源β干扰素(Ser17hI FN-β)的重组腺病毒,体外评价其对结肠癌细胞的抑制增殖作用及细胞周期的影响。方法:人工合成含Ser17突变型hI FN-β基因并克隆至穿梭载体pshuttle-CMV,pshuttle-Ser17hI FNβ与腺病毒骨架质粒pA d同源重组后转染HEK293细胞,包装重组腺病毒rA d-Ser17hI FNβ。rA d-Ser17hI FNβ转染人结肠癌细胞株HT-29,Western blot检测rA d-Ser17hI FNβ在HT-29中的表达;MTT细胞生长实验检测rA d-Ser17hI FNβ对HT-29细胞的体外增殖的影响,流式细胞术检测HT-29细胞周期改变情况。结果:重组腺病毒经HEK293细胞扩增后滴度可达109.125CCID50/ml;Western blot检测到外源性Ser17hI FN-β基因在HT-29细胞中的表达;rA d-Ser17hI FNβ转染后HT-29细胞生长受到抑制(P<0.05),细胞处于S期百分比增加。结论:表达Ser17突变型hI FN-β的重组腺病毒能抑制结肠癌细胞的增殖及诱导S期阻滞,为应用β干扰素对结肠癌进行基因治疗提供了实验基础。
Objective： To investigate the effect of replication-defective recombinant adenovirus expressing Ser^（17） mutant human IFN-β on the cell proliferation and cycle regulation of a human colon carcinoma cell line in vitro. Methods： Ser^（17） mutant human IFN-βgene was inserted into a shuttle plasmid（ pshuttle-CMV）. The pshuttle-Ser^（17） hI FNβ was transformed into Ecoli BJ5183 to make a homologous recombination with adenovirus skeleton genome（ pA deasy）. And the recombinant plasmid pA d-Ser^（17） hI FNβ was transfected into HEK-293 cell to produce mature adenovirus particles. The change of proliferation and cell cycle of rA d infected HT-29 were analyzed by MTT assay and FACS,respectively. Results： The recombinant adenovirus（ rA d-Ser^（17） hI FNβ） with the titer of 109. 125CCID50 /ml was produced. HT29 cells could be infected with the rA d-Ser^（17） hI FNβ in 20 MOI. The expression of Ser^（17） hI FN-β in HT-29 could be detected by Western blot. Significantly,the rA d-Ser^（17）IFNβ inhibited the growth of HT-29 cells and prevented cell cycle progression in S phage in vitro. Conclusion： The recombinant adenovirus expressing Ser^（17）-mutant human IFN-β could suppress the proliferation of colon carcinoma cell in vitro. It provides an experimental basis to apply the IFN-β gene therapy for colon carcinoma in the future.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2017年第2期196-200,205,共6页
Chinese Journal of Immunology
基金
昆明市科技计划重点项目(2015-1-S-00877)
昆明学院人才项目(YJ216002)
昆明学院科学研究项目资助(XJL12026)
