中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
4期
791-793
,共3页
Pokemon蛋白%小干扰RNA%NCI-H520%重组质粒%增殖抑制
Pokemon蛋白%小榦擾RNA%NCI-H520%重組質粒%增殖抑製
Pokemon단백%소간우RNA%NCI-H520%중조질립%증식억제
Pokemon protein%Small interfering RNA%NCI-H520%Recombinant plasmid%Proliferation inhibition
目的 利用RNA干扰(RNAi)技术将针对性构建的重组质粒转染入人肺癌细胞株NCI-H520,观察下调Pokemon基因对人肺癌细胞生物学行为的影响.方法 利用Ambion公司设计软件设计针对Pokemon基因的2条小干扰RNA(siRNA)序列,并合成2对互补的siRNA编码DNA片段,定向克隆至RNAi-Ready pSIREN-RetroQ载体的BamH Ⅰ和EcoR Ⅰ位点,构建重组体质粒,分别命名为pSIREN-Pokemon-1和pSIREN-Pokemon-2,同时设立转染空载体对照组.Western blot检测人肺癌细胞中Pokemon蛋白表达,噻唑蓝(MTT)比色实验检测转染后细胞的增殖抑制结果,流式细胞分析技术检测转染后细胞周期的分布.结果 利用RNAi-Ready pSIRENR RetroQ载体构建的重组质粒可以成功抑制人肺癌细胞NCI-H520中Pokemon蛋白的表达.Western blot检测发现人肺癌细胞NCI-H520中Pokemon蛋白高水平表达,转染后其表达水平明显下降.转染后第3天,MTT比色实验显示pSIREN-Pokemon-1组(0.001 5 ±0.003 0)和pSIREN-Pokemon-2组(0.0086±0.0015)与pSIREN-Pokemon-0组(0.023 0 ±0.001 0)比较差异有统计学意义(P＜0.05),而pSIREN-Pokemon-1与pSIREN-Pokemon-2组之间差异无统计学意义(P＞0.05).流式细胞分析检测发现转染siRNA细胞与转染空载体细胞的细胞周期差异无统计学意义(P＞0.05).pSIREN-Pokemon-1转染组及pSIREN-Pokemon-2转染组形成克隆的数量(18.0±0.3、16.0±0.4)明显低于pSIREN-Pokemon-0组(52.0±0.4,P＜0.05).结论 使用pSIREN载体成功构建的重组质粒转染人肺癌细胞可有效抑制原癌基因Pokemon的表达.转染后细胞增殖受到抑制,生长速度减慢.抑制Pokemon蛋白表达对细胞周期无明显影响.靶向siRNA可以在人肺癌细胞中抑制Pokemon蛋白表达,负调节肺癌细胞生长.
目的 利用RNA榦擾(RNAi)技術將針對性構建的重組質粒轉染入人肺癌細胞株NCI-H520,觀察下調Pokemon基因對人肺癌細胞生物學行為的影響.方法 利用Ambion公司設計軟件設計針對Pokemon基因的2條小榦擾RNA(siRNA)序列,併閤成2對互補的siRNA編碼DNA片段,定嚮剋隆至RNAi-Ready pSIREN-RetroQ載體的BamH Ⅰ和EcoR Ⅰ位點,構建重組體質粒,分彆命名為pSIREN-Pokemon-1和pSIREN-Pokemon-2,同時設立轉染空載體對照組.Western blot檢測人肺癌細胞中Pokemon蛋白錶達,噻唑藍(MTT)比色實驗檢測轉染後細胞的增殖抑製結果,流式細胞分析技術檢測轉染後細胞週期的分佈.結果 利用RNAi-Ready pSIRENR RetroQ載體構建的重組質粒可以成功抑製人肺癌細胞NCI-H520中Pokemon蛋白的錶達.Western blot檢測髮現人肺癌細胞NCI-H520中Pokemon蛋白高水平錶達,轉染後其錶達水平明顯下降.轉染後第3天,MTT比色實驗顯示pSIREN-Pokemon-1組(0.001 5 ±0.003 0)和pSIREN-Pokemon-2組(0.0086±0.0015)與pSIREN-Pokemon-0組(0.023 0 ±0.001 0)比較差異有統計學意義(P＜0.05),而pSIREN-Pokemon-1與pSIREN-Pokemon-2組之間差異無統計學意義(P＞0.05).流式細胞分析檢測髮現轉染siRNA細胞與轉染空載體細胞的細胞週期差異無統計學意義(P＞0.05).pSIREN-Pokemon-1轉染組及pSIREN-Pokemon-2轉染組形成剋隆的數量(18.0±0.3、16.0±0.4)明顯低于pSIREN-Pokemon-0組(52.0±0.4,P＜0.05).結論 使用pSIREN載體成功構建的重組質粒轉染人肺癌細胞可有效抑製原癌基因Pokemon的錶達.轉染後細胞增殖受到抑製,生長速度減慢.抑製Pokemon蛋白錶達對細胞週期無明顯影響.靶嚮siRNA可以在人肺癌細胞中抑製Pokemon蛋白錶達,負調節肺癌細胞生長.
목적 이용RNA간우(RNAi)기술장침대성구건적중조질립전염입인폐암세포주NCI-H520,관찰하조Pokemon기인대인폐암세포생물학행위적영향.방법 이용Ambion공사설계연건설계침대Pokemon기인적2조소간우RNA(siRNA)서렬,병합성2대호보적siRNA편마DNA편단,정향극륭지RNAi-Ready pSIREN-RetroQ재체적BamH Ⅰ화EcoR Ⅰ위점,구건중조체질립,분별명명위pSIREN-Pokemon-1화pSIREN-Pokemon-2,동시설립전염공재체대조조.Western blot검측인폐암세포중Pokemon단백표체,새서람(MTT)비색실험검측전염후세포적증식억제결과,류식세포분석기술검측전염후세포주기적분포.결과 이용RNAi-Ready pSIRENR RetroQ재체구건적중조질립가이성공억제인폐암세포NCI-H520중Pokemon단백적표체.Western blot검측발현인폐암세포NCI-H520중Pokemon단백고수평표체,전염후기표체수평명현하강.전염후제3천,MTT비색실험현시pSIREN-Pokemon-1조(0.001 5 ±0.003 0)화pSIREN-Pokemon-2조(0.0086±0.0015)여pSIREN-Pokemon-0조(0.023 0 ±0.001 0)비교차이유통계학의의(P＜0.05),이pSIREN-Pokemon-1여pSIREN-Pokemon-2조지간차이무통계학의의(P＞0.05).류식세포분석검측발현전염siRNA세포여전염공재체세포적세포주기차이무통계학의의(P＞0.05).pSIREN-Pokemon-1전염조급pSIREN-Pokemon-2전염조형성극륭적수량(18.0±0.3、16.0±0.4)명현저우pSIREN-Pokemon-0조(52.0±0.4,P＜0.05).결론 사용pSIREN재체성공구건적중조질립전염인폐암세포가유효억제원암기인Pokemon적표체.전염후세포증식수도억제,생장속도감만.억제Pokemon단백표체대세포주기무명현영향.파향siRNA가이재인폐암세포중억제Pokemon단백표체,부조절폐암세포생장.
Objective The aim of this research was to observe the influence of the low expression of Pokemom gene to biological behaviour of human lung cancer cell after transfecting the targeted recombinant plasmid into human lung cancer cell line NCI-H520 by using RNA interference (RNAi) technology.Methods Two strips targeted small interfering RNA (siRNA) sequences,which is aimed at the Pokemon gene,were designed by using the design software of Ambion company,and two pairs of complementary DNA fragments of siRNA were also synthesized,then cloned into BamH Ⅰ and EcoR Ⅰ of the RNAi-Ready pSIREN-RetroQ vector,which were named as pSIREN-Pokemon-1 and pSIREN-Pokemon-2,respectively.The control group was set at the mean time.Western blotting was used to test the expression of Pokemon protein,Methyl thiazol tetrazolium (MTT) was to the inhibition of cell proliferation,and flow cytometry was for the cell cycle analysis.Results (1) The recombinant plasmids which were constructed by RNAi-Ready pSIREN-RetroQ vector successfully inhibited the expression of Pokemon protein in human lung cancer cell NCI-H520.The detective results of Western blotting showed the high expression level of Pokemon protein in NCI-H520,but the expression level decreased obviously after transfection.(2) The results of MTT revealed that there was significantly proliferation in pSIREN-Pokemon-1 (0.001 5-± 0.003 0)and pSIREN-Pokemon-2 (0.008 6 ±0.001 5) in comparison with pSIREN-Pokemon-0 (0.023 0 ±0.001 0,P ＜0.05).3 days after transfection,whereas there was no significant difference between pSIREN-Pokemon-1 与 pSIREN-Pokemon-2 (P ＞ 0.05).(3) Flow cytometry analysis showed that there was no significant difference between transfected cells and control group which were transfected blank vector into cells.(4) Cell clone forming experiments suggested that the number and size of clone both reduced compared with control group.Conclusion (1) The recombinant plasmids which were constructed successfully by using pSIREN vector effectively suppressed the expression of proto-oncogene Pokemon.(2) After transfection,the proliferation of NCI-H520 cells was inhibited and the growth was in a lower rate.(3) There were no significantly affects on cell cycle after inhibiting the protein expression of Pokemon.(4) The expression of Pokemon protein could be inhibited after targeting the siRNA.Besides,the growth of lung cancer ceils were negatively regulated.
