摘要
目的构建人微小RNA（miRNA，miR）-637慢病毒过表达载体，建立稳定过表达miR-637的甲状腺乳头癌TPC-1细胞株，检测其对细胞增殖的影响。方法设计针对miR-637前体的过表达基因片段，应用聚合酶链反应法（PCR）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测序和比对分析，构建GV369-miR-637慢病毒表达载体。用慢病毒液感染TPC-1，建立稳定过表达miR-637的TPC-1细胞株。荧光显微镜下观察GV369-miR-637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GV369-miR-637-TPC-1、GV369-NC—TPC-1和TPC-13组细胞中miR-637的表达水平，细胞计数试剂盒（CCK-8）检测各组细胞的增殖能力。结果经限制性内切酶鉴定及DNA测序分析，成功构建GV369-miR-637慢病毒表达载体质粒。GV369-miR-637-TPC-1和GV369-NC—TPC＋1细胞在荧光显微镜下均发出绿色荧光，并且转染效率高。GV369-miR-637-TPC-1组中miR-637的表达水平显著高于GV369-NC—TPC-1细胞组（45．32±3．85比2．78±1．15，P〈0．05），其增殖能力受到抑制。结论成功构建过表达miR-637的TPC-1细胞株，其抑制细胞增殖。
Objective To construct a human microRNA （miRNA, miR） -637 lentiviral expression vector and to establish a stable TPC - 1 cell line of transfected miR - 637, and to detect the effect on cell proliferation. Methods Pri -miR- 637 gene fragment amplified by polymerase chain reaction （PCR）, then construction GV369 -miR -637 lentiviral expression vector by gene recombination technology. Observe GV369 - miR - 637 lentiviral expression vector transfeetion under the fluorescence microscope. The expression levels of miR - 637 were detected in GV369 - miR - 637 - TPC - 1, GV369 - TPC - 1 and TPC - 1 cell lines. The proliferation ability of the cells was detected by cell counting kit - 8 （ CCK - 8 ）. Results We constructed the GV369 -miR -637 slow virus expression vector after the restriction enzyme i- dentification and DNA sequencing analysis. The expression level of miR - 637 in GV369 - miR - 637 - TPC - 1 cell group was significantly higher than that in GV369 - NC - TPC -1 cell group （45.32± 3.85 vs. 2.78 ± 1.15, P 〈 0. 05 ）, and the ability of proliferation was inhibited. Conclusion We successfully constructed the miR - 637 lentiviral expression vector and the TPC - 1 - miR - 637 cell line. We successfully constructed the TPC - 1 cell line of expressing miR - 637, which inhibited cell proliferation.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第7期1762-1764,共3页
Chinese Journal of Experimental Surgery
基金
河南省医学科技攻关计划项目（201402007）
