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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。17
OBJECTIVE: To construct a recombinant adenovirus expressing mutant human interferon beta (Ser17hI FN-β) and evaluate its effect on proliferation and cell cycle inhibition of colon cancer cells in vitro. Methods: The recombinant plasmid hI FN-β containing ser17 was synthesized and cloned into shuttle vector pshuttle-CMV. Pshuttle-Ser17hI FNβ was homologously recombined with adenovirus backbone plasmid pA d and transfected into HEK293 cells. The recombinant adenovirus rA d-Ser17hI FNβ. rA d-Ser17hI FNβ was transfected into human colon cancer cell line HT-29. The expression of rA d-Ser17hI FNβ in HT-29 cells was detected by Western blot. MTT cell growth assay was used to detect the proliferation of HT-29 cells induced by rA d-Ser17hI FNβ Flow cytometry was used to detect the cell cycle changes in HT-29 cells. Results: The titer of recombinant adenovirus was 109.125CCID50 / ml after amplification by HEK293 cells. The expression of exogenous Ser17hI FN-β gene in HT-29 cells was detected by Western blot. After transfection with rA d-Ser17hI FNβ -29 cell growth was inhibited (P <0.05), the percentage of cells in the S phase increased. CONCLUSION: Recombinant adenovirus expressing Ser17 mutant hI FN-β can inhibit the proliferation and induce the S phase arrest in colon cancer cells. It provides an experimental basis for gene therapy of colon cancer with interferon-beta. 17