Ad.RGD-ING4和PTEN对白血病细胞株抑癌作用  

Transfection efficiency and tumor suppressor effect of Ad.RGD-PTEN and ING4 on leukemia cell

作　　者：韩亚丽 王家融[2] 杨吉成[2] 盛伟华[2] 缪竞诚[2] 

机构地区：[1]上海交通大学医学院附属上海儿童医学中心血液肿瘤科,上海200127 [2]苏州大学医学部细胞与分子生物学教研室,江苏苏州215123

出　　处：《内科理论与实践》2016年第2期95-102,共8页Journal of Internal Medicine Concepts & Practice

基　　金：国家自然科学基金青年科学基金(项目编号:81001016)

摘　　要：目的：构建RGD修饰的生长抑制因子4（ING4）和（或）10号染色体缺失的磷酸酶及张力蛋白同源的基因（PTEN）单、双基因重组腺病毒载体,以研究其对白血病细胞株的感染效率和抑瘤作用的可行性。方法：在本科室已成功构建的p Ad Track-巨细胞病毒（CMV）-多腺苷酸（poly A）启动子腺病毒载体上,通过常规的分子生物学方法,获得RGD修饰的ING4和（或）PTEN单、双基因重组腺病毒,将其与未经RGD修饰的基因重组腺病毒同时感染QBI-293A细胞,检测和比较病毒的效价,并以10、50、100、200感染复数（MOI）不同剂量感染不同肿瘤细胞和人胚肺正常二倍体细胞系WI-38,培养48 h后在荧光显微镜下观察和用流式细胞仪检测,通过比较各组细胞中绿色荧光蛋白（GFP）阳性细胞比例来检测其对不同肿瘤细胞的感染效率。结果：经荧光显微镜观察各组重组腺病毒可见GFP的表达,反转录（RT）-PCR鉴定结果表明均可产生预期大小750 bp的ING4和1 209 bp的PTEN PCR产物,基因测序结果与预期序列相一致。RGD修饰的基因重组腺病毒载体效价分别比未经修饰的基因重组腺病毒效价高3～4倍（P=0.027）。流式细胞仪检测显示RGD修饰的基因重组腺病毒比未经修饰的基因重组腺病毒载体对各组肿瘤靶细胞有更高的GFP表达,感染效率明显提高,尤其对人白血病细胞系K562、Thp-1、MEG-01差异最为明显,未经改造的腺病毒载体感染白血病细胞后GFP阳性率仅3.16%～5.01%,而带有RGD的腺病毒载体感染后GFP阳性率可达到23.11%～31.58%（P=0.001 3）。结论 ：成功构建了RGD修饰的ING4和（或）PTEN单、双基因重组腺病毒载体。RGD修饰的ING4和（或）PTEN基因重组腺病毒明显提高了腺病毒的效价,在各种肿瘤细胞感染效率中以白血病细胞系K562、Thp-1、MEG-01最为明显。Objective To study the transfection efficiency and tumor-suppressing effect of the constructed Ad.RGD inhibitor of growth protein-4（ING-4） and/or phosphatase and tensin homolog deleted on chromosome ten（PTEN）recombinant viral vector on leukemia cell.Methods ING4 and PTEN fragments were subcloned into p Ad Trackcytomegaoviyns（CMV）-poly A-promoter to form p Ad Track-CMV-ING4-poly A-promoter,p Ad Track-CMV-poly A-promoterPTEN and p Ad Track-CMV-ING4-poly A-promoter-PTEN transfer vectors,respectively.The transfer vectors linearized with Pme I digestion and p Ad.RGD backbone vector were further co-transformed for homologous recombination,then transfected into QBI-293 A cells to obtain Ad.RGD-ING4,Ad.RGD-PTEN and Ad.RGD-ING4-PTEN.U87,U251,WI-38,MCF-7,A549,HT29,K562,MEG-01 and THP-1 cells were transfected with Ad.RGD-ING4,Ad.RGD-PTEN,Ad.RGDING4-PTEN,Ad.RGD,Ad.ING4,Ad.PTEN,Ad.ING4-PTEN and adenovirus for 48 h at 10,50,100 and 200 multiplicity of infections（MOIs）,respectively.Efficiency of tumor cell transfection was evaluated by number of green fluorescent protein（GFP） positive cells under fluorescence microscopy and flow cytometry（FCM）.Results Seven hundred and fifty bp ING4 and 1 209 bp PTEN polymerase chain reaction（PCR） products were detected by reverse transcription（RT）-PCR.The results of gene sequencing were consistent with the expected sequences.GFP under fluorescence microscopy denoted a significant expression in all kinds of tumor cells transfected.The transfection efficiency of adenovirus vector modified by RGD was 3-4 times of adenovirus vector without RGD in all kinds of above-mentioned tumor cells（P=0.027）.Flow cytometry showed that GFP expression in all kinds of tumor cells transfected by adenovirus vector modified with RGD were higher than the unmodified,especially in leukemia cell line K562,Thp-1 and MEG-01,in which GFP positive cell rates were 23.11%-31.58% vs 3.16%-5.01%（P =0.001 3）.Conclusions Ad.RGD-ING4,Ad.RGD-PTEN and Ad.RGD-ING4-PTEN were successfully constru

关 键 词：RGD 白血病细胞株 生长抑制因子4 10号染色体缺失的磷酸酶及张力蛋白同源的基因 基因治疗 

分 类 号：R733.7[医药卫生—肿瘤] R392.11[医药卫生—临床医学]