过表达缺氧诱导因子-1α的结直肠癌裸鼠可视化模型的建立  

Establishment of a nude mouse model of fluorescently tagged-colorectal cancer overexpressing hypoxia-inducible factor-1α

作　　者：李喆[1] 叶小磊 杨生生[3] 殷佩浩[4] 胡送娇[4] 陈磊[1] 房林[1] 

机构地区：[1]同济大学附属第十人民医院甲状腺乳腺外科,上海200072 [2]宁波市医学科学研究所药物与药理研究室,浙江宁波315020 [3]第二军医大学生物化学与分子生物学教研室,上海200433 [4]上海中医药大学附属普陀中心医院普通外科,上海200062

出　　处：《中国肿瘤生物治疗杂志》2015年第2期246-251,共6页Chinese Journal of Cancer Biotherapy

基　　金：上海市科学技术委员会专项基金资助项目(No.12140902302)~~

摘　　要：目的:建立一种荧光可视化显像且稳定过表达缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)的裸鼠结直肠癌移植瘤模型。方法:对不同的人结直癌细胞系(SW480、SW620、LOVO及HCT116细胞)用缺氧诱导剂Co Cl2处理,根据HIF-1α被诱导表达的情况选择合适的靶细胞;将HIF-1α的c DNA序列克隆至慢病毒表达质粒p LV-TRC-EGFP,构建p LVHIF1α-EGFP慢病毒表达质粒,并包装过表达HIF-1α的慢病毒颗粒Lenti-HIF1α-EGFP。将Lenti-HIF1α-EGFP感染SW480细胞,嘌呤霉素筛选稳定过表达HIF-1α的细胞株SW480HIF-1α,Western blotting检测SW480HIF-1α细胞内HIF-1α及其下游蛋白VEGF、M1型丙酮酸激酶(pyruvate kinase expression M1,PKM1)的表达情况,Transwell实验检测其迁移能力。裸鼠腹腔注射SW480HIF-1α建立结直肠癌移植瘤模型,倒置荧光显微镜观察裸鼠腹腔移植瘤结节。结果:常氧条件下4种结直肠癌细胞均不表达HIF-1α,经Co Cl2诱导后,除SW480细胞系以外的细胞均可被诱导表达HIF-1α,故选择SW480细胞作为研究靶细胞。成功构建稳定过表达HIF-1α的细胞株SW480HIF-1α,SW480HIF-1α细胞内HIF-1α、VEGF和PKM1的表达水平均高于野生型SW480细胞,其迁移能力较野生型SW480细胞显著增强[观察视野内迁移细胞数:(250±11)vs(50±5)个,P<0.01]。SW480HIF-1α组裸鼠肠壁形成肿瘤结节数量显著多于SW480组[(15±4)vs(4±1)个,P<0.05]。结论:成功建立了高表达HIF-1α的荧光可视化裸鼠移植瘤模型,为进一步的相关功能学研究以及药物筛选提供条件。Objective:To establish a nude mice xenograft model of fluorescently tagged-colorectal cancer overexpression of hypoxia-inducible factor-1α( HIF-1α). Methods: Four human colorectal cancer cell lines,SW480,SW620,LOVO and HCT116,were treated with Co Cl2,and the one that remained HIF-1α-negative under Co Cl2 induction was chosen.The selected cell line was infected with a lentiviral vector overexpressing HIF-1α and EGFP,Lenti-HIF1α-EGFP. Infected cells underwent puromycin selection. Cells stably expressing HIF-1α and EGFP. HIF-1α,confirmed by Western blotting,were used in transwell assay of migration in vitro and injected intraperitoneally into nude mice to create a xenograft model of colorectal cancer in vivo. Results: After Co Cl2 induction,only SW480 remained HIF-1α-negative and was thus selected. SW480 cells stably overexpressing HIF-1α and EGFP had enhanced migration ability(250 ± 11) as compared with control Sw480 cells(50 ± 5)( P < 0. 01). The number of tumor nodules formed on the abdominal wall was significantly higher in mice injected with SW480 cells overexpressing HIF-1α(15 ± 4) than in mice injected with control cells(4 ± 1)(P <0. 05). Conclusion: Injection of endogenously HIF-1α-negative colorectal cancer SW480 cells infected by a lentiviral vector overexpressing HIF-1α and EGFP into nude mice would provide a feasible approach of creating a xenograft model of fluorescently visible colorectal cancer for investigations on the function of HIF-1α in the pathogenesis of colorectal cancer.

关 键 词：结直肠癌 移植瘤模型 稳定转染 慢病毒 缺氧诱导因子-1Α 绿色荧光蛋白 

分 类 号：R735.34[医药卫生—肿瘤] R-332[医药卫生—临床医学]