稳定过表达miR-224的胰腺黏液性囊腺癌MCC1细胞株的建立  

Establishment of pancreatic mucinous cystadenocarcinoma cell line MCC1 with stable overexpression of miR-224

作　　者：彭小波[1] 郭承涛 应明真[1] 李洁[1] 宋乐乐 吴燕君 詹丽杏 湛先保[1] PENG Xiaobo;GUO Chengtao;YING Mingzhen;LI Jie;SONG Lele;WU Yanjun;ZHAN Lixing;ZHAN Xianbao(Department of Oncology,Changhai Hospital,Second Military Medical University,Shanghai,200433,China;Institute for Nutritional Science,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai,200031,China)

机构地区：[1]第二军医大学长海医院肿瘤科,上海200433 [2]中国科学院上海生命科学研究院营养所,上海200031

出　　处：《中国肿瘤生物治疗杂志》2018年第7期721-725,共5页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81672892)~~

摘　　要：目的:构建hsa-micro RNA-224(miR-224)过表达慢病毒载体,建立稳定过表达miR-224的胰腺黏液性囊腺癌MCC1细胞株。方法:设计miR-224前体过表达基因片段,应用q RT-PCR法扩增目的基因片段,通过基因重组技术将目的基因片段插入GV369慢病毒载体中,进行PCR鉴定及DNA测序比对分析。用GV369-miR-224慢病毒感染胰腺黏液性囊腺癌MCC1细胞,建立稳定过表达miR-224的MCC1细胞株。荧光显微镜下观察GV369-NC及GV369-miR-224慢病毒表达载体的转染效果,反转录-聚合酶链反应(RT-PCR)检测MCC1、GV369-miR-224-MCC1和GV369-NC-MCC1组细胞中miR-224的表达水平。结果:成功构建GV369-miR-224慢病毒表达载体质粒。GV369-miR-224-MCC1、GV369-NC-MCC1细胞在荧光显微镜下均发出绿色荧光。miR-224表达水平在GV369-miR-224-MCC1细胞中显著高于阴性对照GV369-NC-MCC1细胞和空白对照MCC1细胞(23.45±1.94 vs2.11±0.38,1.46±0.11,均P<0.01),而两组对照组之间miR-224表达水平差异无统计学意义(P>0.05)。结论:成功构建稳定过表达miR-224的胰腺黏液性囊腺癌MCC1细胞株,为探讨miR-224在胰腺黏液性囊腺癌中的功能及发病机制提供新的细胞模型。Objective： To construct a hsa-microRNA-224（miR-224） lentiviral expression vector and to establish pancreatic mucinous cystadenocarcinoma MCC1 cell line with stable miR-224 over-expression. Methods： Pri-miR-224 gene fragment was designed and amplified by quantitative real-time polymerase chain reaction （qRT-PCR）, and then loaded into GV369 lentiviral vectors （GV369-miR-224） by gene recombination technology. GV369-miR-224 lentivrial expression vectors were then identified by PCR and DNA sequencing.The GV369-miR-224 vector fluid was then used to infect pancreatic mucinous cystadenocarcinoma MCC1 cell line to establish the MCC1 cell line stably over-expressing miR-224. The transfection efficiency of GV369-NC and GV369-miR-224 was observed under fluorescence microscopy; and the expression levels of miR-224 in MCC1, GV369-miR-224-MCC1 and GV369-NC-MCC1 cell lines were detected by RT-PCR. Results： The GV369-miR-224 lentiviral vectors were successfully constructed. GV369-miR-224-MCC1 and GV369-NC-MCC1 cells all emit green fluorescence under the fluorescence microscope. The expression level of miR-224 in GV369-miR-224-MCC1 cell group was significantly higher than that in negative control GV369-miR-224-MCC1 group and blank control MCC1 cell group （23.45±1.94,1.46±0.1 and 2.11±0.38, P〈0.01）, however, there was no significant difference between the two control groups （P〉0.05）. Conclusion： A pancreatic mucinous cystadenocarcinoma MCC1 cell line with stable miR-224 over-expression was successfully established, and this will provide a new cell model for exploring the function and pathogenesis of miR-224 in pancreatic mucinous cystadenocarcinoma.

关 键 词：miR-224 慢病毒载体 胰腺黏液性囊腺癌细胞株 

分 类 号：R735.9[医药卫生—肿瘤] R733[医药卫生—临床医学]