目的：构建hsa-microRNA-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qRT-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 vs 2.11±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.

国家自然科学基金资助项目(No. 81672892)