摘要
目的构建人IL-24的腺病毒载体Ad-IL24,观察Ad-IL24对HL-60细胞体外培养的影响。方法以pcDNA3.0-IL24重组质粒为模板PCR扩增IL-24基因,酶切并连接到带有绿色荧光蛋白(GFP)标记的pAdTrack-CMV质粒上,Pme I线性化重组质粒pAdTrack-CMV-IL24,并与腺病毒质粒pAdEasy-1共转化BJ5183细菌,获得重组腺病毒载体pAdEasy-1-pTrack-CMV-IL24,经Pac I线性化后转染QBI-293A细胞,收获重组病毒Ad-IL24。将它感染HL-60细胞,通过激光扫描共聚焦显微镜(ISCM)、MTT和流式细胞术(FCM)法检测IL-24对HL-60细胞生长的影响,免疫细胞化学分析凋亡因子的改变,ELISA检测重组病毒对HL-60细胞分泌γ干扰素(IFN-γ)和肿瘤坏死因子-α(TNF-α)的影响。结果成功构建重组腺病毒载体pAdEasy-1-pTrack-CMV-IL24并获得高滴度的重组腺病毒Ad-IL24,各种检测方法表明IL-24基因能抑制HL-60细胞生长,诱导凋亡,IL-24还能下调HL-60细胞Bcl-2的表达并刺激它分泌二级细胞因子IFN-γ和TNF-α。结论IL-24能抑制HL-60细胞生长、诱导凋亡,它可通过下调HL-60细胞Bcl-2的表达并刺激其分泌具有抗肿瘤活性的二级细胞因子来发挥效应。
Objective To construct the human IL-24 recombined adenovirus (Ad-IL24) and to observe the effect of Ad-IL24 on ex vivo culture of HL-60 cells. Methods pAdTrack-CMV-IL24 was con- strutted by PCR with pcDNA3.0-IL24 recombined plasmid as a template, enzyme digestion and ligation. The pAdTrack-CMV-IL24 lineared by Pme I was co-transformed into BJ5183 with pAdEasy-1. The pAdEasy-1-pTrack-CMV-IL24 recombined adenovirus vector was lineared with Pac I and then transfected in-to QBI-293A cells. The Ad-IL24 was obtained and used to infect HL-60 cells, and the effect on HL-60 cells was tested by LSCM, MTF, FCM, ELISA and immunohistochemistry staining. Results The pAdEasy-1- pTrack-CMV-IL24 vector was constructed and the Ad-IL24 was successfully obtained. The effect of inhibiting growth of HL-60 cells and inducing cell apoptosis by IL-24 was proved by LSCM, MTT and FCM. IL-24 down-regulated the expression of anti-apoptosis factor Bcl-2 and increased the secretion of IFN-γ, TNF-α of HL-60 cells. Conclusion Ad-IL24 can inhibit the growth of HL-60 cells and induce cell apoptosis through down-regulating expression of anti-apoptosis factor and increasing the secretion of IFN-γ, TNF-α of HL-60 cells. The human Ad-IL24 may provide a basic study for the future target therapy to tumors.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2008年第4期325-329,共5页
Chinese Journal of Microbiology and Immunology
基金
基金项目:无锡市社会发展科技计划项目(CSZ00730)
苏州大学医学发展基金项目(EE134517)