CAJ | 학술논문

目的:建立RNAi抑制柯萨奇病毒腺病毒受体(coxsackievirus and adenovirus receptor,CAR)基因表达的肺鳞状细胞癌NCI-H520稳定表达细胞系,将CAR基因沉默的NCI-H520细胞移植于裸鼠体内构建移植瘤模型,观察裸鼠体内成瘤率及对瘤体生长等方面的影响.方法:将针对CAR mRNA序列设计的小干扰RNA重组质粒分别以脂质体转染至NCI-H520细胞,并经G418抗性筛选得到稳定细胞系;用平板克隆形成实验观察RNAi抑制CAR基因表达对NCI-H520细胞增殖的影响;通过动态观察瘤体的生长,进一步验证CAR对肺癌的增殖是否有促进作用.结果:筛选出抑制CAR基因表达效果最佳的一组shRNA,并建立了能稳定而有效抑制CAR基因表达的肺鳞癌NCI-H520细胞系.平板克隆形成实验显示,shRNA-2抑制CAR基因表达后NCI-H520细胞的增殖能力有所下降,但其差异无统计学意义(P>0.05);且在肿瘤形成实验中,实验组瘤重显著低于对照组(P<0.01).结论:裸鼠肺癌移植瘤模型构建成功,为今后肺癌的研究奠定基础.利用RNAi有效抑制了CAR基因的表达,并抑制NCI-H520细胞在裸鼠体内瘤体的生长,CAR有望成为肺癌基因治疗的靶点之一.
목적:건립RNAi억제가살기병독선병독수체(coxsackievirus and adenovirus receptor,CAR)기인표체적폐린상세포암NCI-H520은정표체세포계,장CAR기인침묵적NCI-H520세포이식우라서체내구건이식류모형,관찰라서체내성류솔급대류체생장등방면적영향.방법:장침대CAR mRNA서렬설계적소간우RNA중조질립분별이지질체전염지NCI-H520세포,병경G418항성사선득도은정세포계;용평판극륭형성실험관찰RNAi억제CAR기인표체대NCI-H520세포증식적영향;통과동태관찰류체적생장,진일보험증CAR대폐암적증식시부유촉진작용.결과:사선출억제CAR기인표체효과최가적일조shRNA,병건립료능은정이유효억제CAR기인표체적폐린암NCI-H520세포계.평판극륭형성실험현시,shRNA-2억제CAR기인표체후NCI-H520세포적증식능력유소하강,단기차이무통계학의의(P>0.05);차재종류형성실험중,실험조류중현저저우대조조(P<0.01).결론:라서폐암이식류모형구건성공,위금후폐암적연구전정기출.이용RNAi유효억제료CAR기인적표체,병억제NCI-H520세포재라서체내류체적생장,CAR유망성위폐암기인치료적파점지일.
This work aimed to establish a lung squamous carcinoma cell line in which the coxsackievirus and adeno-virus receptor (CAR) gene expression is stably suppressed by ribonucleic acid (RNA) interference. Colony formation experiments and orthotopic tumor model building were employed to detect the correlation between CAR and tumor formation. Methods:Three small in-terfering RNAs (siRNA) targeting the CAR gene were transfected into a lung squamous carcinoma NCI-H520 cell line. G418 was used to select a cell line where CAR gene expression was stably suppressed by RNA interference. The expression levels of CAR mRNA and protein were detected by Western blot in three NCI-H520 cell lines. The cell line where CAR gene expression was most suppressed was selected. Experiments on colony formation and tumorigenesis were employed to determine the effects of CAR gene on the proliferation of NCI-H520 cells after the CAR expression was reduced. Results:Three NCI-H520 cell lines wherein CAR gene expression was sta-bly suppressed were obtained. Results of the colony formation assays suggested that the reduced CAR levels were associated with weak-ened cell proliferation. However, these data did not reach a statistical significance. The CAR-silenced NCI-H520 clones (CAR-) were inhibited in their ability to engraft the virocytes into the nude mice. Conclusions:A cell line NCI-H520 in which CAR gene expression was stably and efficiently suppressed was successfully constructed. The RNAi-mediated gene silencing of CAR can significantly affect the growth of NCI-H520 cells in the tumor-bearing nude mice. CAR is expected to become a new target for treating lung cancers.