摘要
目的 针对肺癌相关基因SLC35F2构建RNA干扰（RNAi）重组慢病毒质粒并进行慢病毒包装,建立SLC35F2表达稳定抑制的H1299肺癌细胞株,并探讨SLC35F2基因的功能.方法 应用pGCSIL-PUR慢病毒载体构建针对SLC35F2的ShRNA载体,转染包装293T细胞,收集病毒上清转染肺癌细胞株H1299,经嘌呤霉素（puromycin）筛选并扩大培养得到稳定克隆;实时荧光定量聚合酶链反应（Real-time PCR）和Western blot检测癌细胞内SLC35F2的表达;CCK-8比色法检测细胞增殖;Annexin V-FITC/PI双染法检测细胞凋亡.结果 成功构建SLC35F2-ShRNA慢病毒载体（SLC-siRNA）及SLC35F2表达稳定抑制的肺癌细胞株,经过检测证实SLC-siRNA慢病毒载体对SLC35F2的抑制效率达81.8%;CCK-8检测显示SLC-siRNA稳定转染的H1299细胞株生长抑制率达16.3%,稳定转染株凋亡细胞百分比较阴性对照组明显升高（14.88%比3.16%,P＜0.05）.结论 慢病毒载体介导的靶向SLC35F2的RNAi可有效抑制SLC35F2表达,降低肺癌细胞的增殖能力,增加其凋亡比例.
Objective To investigate the possibility of SLC35F2 inhibition by lentiviral vector-mediated RNA interference （RNAi） and the influence on cell proliferation and apoptosis in lung cancer cell line,and to set up a lung cancer cell line in which SLC35F2 is stably suppressed.Methods The lentiviral vector of siRNA targeted against SLC35F2 （SLC-siRNA） was constructed and transfected into the packaging cells 293T,and the viral supernatant was collected to transfect H1299 cells.After selection by puromycin and culture expansion,the stable cell clones were attained.Quantitative real-time fluorescent polymerase chain reaction （PCR） and Western blotting were used to detect the expression of SLC35F2.The effect of SLC35F2 silencing by RNAi on cell proliferation was quantified by CCK-8 assay.Annexin V-FITC/PI staining was employed to examine the apoptosis.Results Lentiviral vector SLC35F2-shRNA was constructed successfully.As compared with control group,the SLC35F2 expression was decreased to 81.8% in RNA and protein levels.CCK-8 revealed that the inhibition rate of H1299 cells transfected with SLC35F2 was 16.3%,and the apoptosis rate was significantly increased as compared with negative control group （ 14.88% vs 3.16% ,P 〈0.05 ）.Conclusion Lentiviral vector-mediated RNA interference targeting against SLC35F2 can effectively inhibit SLC35F2 expression and cell proliferation.The lung cell line in which SLC35F2 gene was stably suppressed was successfully established.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2011年第3期419-421,F0004,共4页
Chinese Journal of Experimental Surgery
基金
基金项目：国家自然科学基金资助项且（30772486）
高等学校博士学科点专项科研基金资助项目（20070001713）
关键词
肺癌
RNAI
慢病毒载体
细胞增殖
Long carcinoma
RNA interference
Lentiviral vector
Cell proliferation
