肝癌胰岛素样生长因子-I受体异常表达及其转录干预对裸鼠移植瘤的抑制效果  

Abnormal expression of insulin-like growth factor-I receptor and inhibitory effect of its transcription intervention on nude mice xenograft tumor

作　　者：姚敏[1] 严晓娣[1] 蔡胤[1] 顾娟娟[1] 杨绪莉 潘刘翃 王理[1] 姚登福[1] 

机构地区：[1]南通大学附属医院临床医学研究中心医学院免疫教研室,江苏省南通市226001

出　　处：《中华肝脏病杂志》2016年第11期846-851,共6页Chinese Journal of Hepatology

基　　金：国家国际科技合作项目（2013DFA32150）;南通市科技项目（HS2014078）

摘　　要：目的分析肝癌胰岛素样生长因子-I受体（IGF—IR）表达并观察干预基因转录对裸鼠移植瘤的抑制作用。方法对40例原发性肝癌患者，手术后分别留取癌灶、癌周（距癌灶边缘2cm）和远癌肝组织（距癌灶边缘5cm）各40份，约200mg，除部分作病理学检查外，其余置-85℃保存。4～6周龄体质量（18～20）gBALB／C裸鼠18只，雌雄各半，随机分为对照组6只、阴性对照组6只、干扰组6只，于无特定病原体条件下饲养。细胞株以含10％胎牛血清的二甲基亚砜完全培养基于37℃含CO2培养箱培养。细胞接种后，融合度达90％时，将shRNA分为干扰组、阴性对照和未处理对照组，以PolyJet TM转染试剂转染至肝癌细胞，经G418筛选，得到稳定转染细胞克隆，用于体内研究。用免疫组织化学法，Westernblot或荧光定量PCR分析人肝癌组织、肝癌细胞株IGF—IR表达I构建IGF-IR短发夹型RNA（shRNA）真核表达质粒并筛选最有效序列；转染至肝癌PLC／PRF／5细胞，CCK-8法分析细胞增殖变化；经G418筛选稳定株接种裸鼠皮下成瘤，绘制肿瘤生长曲线及组织学检查。统计数据用GraphpadPrism5．0和SPSSl8．0软件进行绘图和分析，多个样本均数间比较先行方差检验和口检验，两样本均数比较采用t检验，样本率间比较采用x2分析或Fisher确切概率法，表达强度以等级相关分析。结果IGF—IR阳性率肝癌组为82．5％、癌周组为42．5％、远癌组为10．0％，癌周组、远癌组与肝癌组比较，x2=13．653和X2=42．290，P值均〈0．01；其表达强度，肝癌组显著高于癌周组（Z=4．771，P〈0．01）和远癌组（Z=6．579，P〈0．01）。肝L02细胞未见IGF—IR明显表达，肝癌PLC／PRF／5细胞IGF-IR呈强表达，约为Bel-7404细胞的4倍、HepG2细胞的5倍；以干扰效果最佳的shRNA4转染PLC／PRF／5细胞，在72h时癌细胞生长平均抑制率为63．9％，IGF-IR在转录水平Objective To investigate the expression of insulin-like growth factor-I receptor （IGF- IR） in liver cancer and the inhibitory effect of its transcription intervention on nude mice xenograft tumor. Methods A total of 40 patients with primaxy liver cancer were enrolled, and 40 samples of cancer lesions, peri-cancerous tissues （with a distance of 2 cm to the margin of cancer lesion）, or distal liver tissues （with a distance of 5 cm to the margin of cancer lesion）, with a weight of 200 mg, were collected after surgery. Some of these samples were used for pathological examination, and the rest were stored at -85℃. A total of 18 BALB/ c nude mice aged 4-6 weeks with a body weight of 18-20 g （9 male and 9 female mice） were randomly divided into control group, negative control group, and co-intervention group, with 6 mice in each group, and fed under specific pathogen-free conditions. The cell line was cultured in the dimethyl sulfoxide complete medium containing 10% fetal bovine serum in a CO2 incubator at 37℃. When the cell confluence reached 90% after cell inoculation, shRNA was divided into co-intervention group, negative control group, and untreated control group and were transfected to hepatoma cells using PolyJetTM transfection reagent. Stable cell clones obtained by （3418 screening and used for the in vivo study. Immunohistochemistry, Westem blotting, and quantitative real-time PCR were used to analyze the expression of IGF-IR in the human hepatoma tissue and cell line. The IGF-IR shRNA eukaryotic expression plasmids were established and screened for the most effective sequence; they were transfected to PLC/PRF/5 hepatoma cells, and the CCK-8 assay was used to analyze the changes in cell proliferation. The stable cell line screened out by G418 was inoculated to establish the subcutaneous xenograft turnor in nude mice. The tumor growth curve was plotted and histological examination was performed. Graphpad Prism 5.0 and SPSS 18.0 were used for plotting and data analysis; the variance test and

关 键 词：癌 肝细胞 IGF-I型受体 基因沉默 

分 类 号：R735.7[医药卫生—肿瘤]