CAJ | 학술논문

目的建立慢病毒介导的靶向干扰电压门控氯通道3(voltage-gated chloride channel 3,ClC-3)的稳定肝癌细胞株,并探讨其侵袭和迁移能力的改变。方法构建靶向 ClC-3的3个短发夹状RNA ( short-hairpin RNA, shRNA)慢病毒表达载体,293FT细胞包装慢病毒并测定滴度。重组慢病毒感染肝癌细胞株MHCC97H,筛选稳定干扰细胞株。实时荧光定量PCR和Western blot检测ClC-3 mRNA和蛋白的表达以确定干扰效率。 Transwell小室实验(含Matrigel和不含Ma-trigel)和细胞划痕实验检测ClC-3基因被抑制后侵袭和迁移能力的改变。结果成功获得4种慢病毒,感染MHCC97细胞后,建立1株阴性对照细胞和3株 ClC-3稳定干扰细胞( MHCC97H/shClC-3-1、shClC-3-2和 shClC-3-3)。3株稳定干扰细胞ClC-3 mRNA和蛋白表达水平明显低于阴性对照细胞(P<0．01),MHCC97H/shClC-3-2细胞对ClC-3的抑制效果最好。与阴性对照细胞相比,MHCC97H/shClC-3-2细胞侵袭和迁移能力都下降( P<0．01)。结论成功建立稳定干扰ClC-3基因的肝癌细胞株,ClC-3表达抑制会降低MH-CC97H细胞的侵袭和迁移能力。
목적건립만병독개도적파향간우전압문공록통도3(voltage-gated chloride channel 3,ClC-3)적은정간암세포주,병탐토기침습화천이능력적개변。방법구건파향 ClC-3적3개단발협상RNA ( short-hairpin RNA, shRNA)만병독표체재체,293FT세포포장만병독병측정적도。중조만병독감염간암세포주MHCC97H,사선은정간우세포주。실시형광정량PCR화Western blot검측ClC-3 mRNA화단백적표체이학정간우효솔。 Transwell소실실험(함Matrigel화불함Ma-trigel)화세포화흔실험검측ClC-3기인피억제후침습화천이능력적개변。결과성공획득4충만병독,감염MHCC97세포후,건립1주음성대조세포화3주 ClC-3은정간우세포( MHCC97H/shClC-3-1、shClC-3-2화 shClC-3-3)。3주은정간우세포ClC-3 mRNA화단백표체수평명현저우음성대조세포(P<0．01),MHCC97H/shClC-3-2세포대ClC-3적억제효과최호。여음성대조세포상비,MHCC97H/shClC-3-2세포침습화천이능력도하강( P<0．01)。결론성공건립은정간우ClC-3기인적간암세포주,ClC-3표체억제회강저MH-CC97H세포적침습화천이능력。
Aim To establish a hepatic carcinoma cell line with stable voltage-gated chloride channel 3 ( ClC-3 ) gene silencing through the lentivirus-mediated short-hairpin RNA ( shRNA ) method and investigate the effects of gene silencing on invasion and migration. Methods Three lentiviral vectors coding shRNA tar-geting ClC-3 gene were constructed, the recombinant plasmids were packaged into mature lentivirus by 293FT cells, and then the lentiviruses were harvested, concentrated and titrated. MHCC97H cells were infec-ted with the recombinant lentiviruses and then were se-lected to obtain cell lines stably expressing ClC-3 shR-NA. The efficiency of ClC-3 mRNA and protein ex-pression interference were determined by real-time flu-orescence quantitative PCR and Western blot, respec-tively. The effects of ClC-3 gene interference on inva-sion and migration of MHCC97 H cells were performed by Transwell chamber assays with or without Matrigel and cell scratch assay. Results The recombinant lentiviral vectors were successfully constructed and four lentiviruses were acquired after packaged by 293 FT cells. One negative control cell line and three cell lines with ClC-3 gene interference ( MHCC97 H/shClC-3-1 , shClC-3-2 and shClC-3-3 ) were successfully construc-ted after MHCC97 H cells were infected with lentivirus-es. The expression level of ClC-3 mRNA and protein in three ClC-3-silenced cells were obviously lower than the negative control cells ( P <0. 01 ) , MHCC97 H/shClC-3-2 cells showed the greatest inhibition of ClC-3 mRNA and protein expressions. As compared with the negative control cells, the ClC-3 gene interference sig-nificantly decreased invasion and migration of MH-CC97 H cells in vitro ( P <0. 01 ) . Conclusion The hepatic carcinoma cell lines with stable ClC-3 gene si-lencing were successfully established and the ClC-3 gene interference could significantly inhibit invasion and migration of MHCC97H cells.