中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
12期
2503-2506
,共4页
孙念峰%田爱玲%徐伯栋%田玉玲%胡三元
孫唸峰%田愛玲%徐伯棟%田玉玲%鬍三元
손념봉%전애령%서백동%전옥령%호삼원
RNA干扰%Bag-1基因%基因治疗%结肠癌
RNA榦擾%Bag-1基因%基因治療%結腸癌
RNA간우%Bag-1기인%기인치료%결장암
RNA interference%Bag-1 gene%Gene therapy%Colon cancer
目的 构建Bag-1短发卡RNA(shRNA)干扰载体并通过内源性筛靶实验检测Bag-1基因mRNA和蛋白的表达,筛选出最佳的干扰靶序列.方法 应用干扰技术构建Bag-1 shRNA干扰载体,在细胞转染预实验中将构建好的带有绿色荧光蛋白的质粒转染Lovo细胞,通过观察绿色荧光蛋白来判断转染的效率,分别应用逆转录-聚合酶链反应(RT-PCR)和Western blot法检测Bag-1在LoVo细胞中的表达.在shRNA干扰序列筛选实验中,通过定量PCR和Western blot法来检测构建的干扰载体在LoVo细胞中的表达.结果 重组质粒分别经双酶切分析,琼脂糖凝胶电泳鉴定可见清晰地切出与Bag-1及pGPHl/GFP/Neo载体大小相符的片段并与DNA Marker相符.用带绿色荧光基因的pGPH1/GFP/Neo-shNC质粒转染LoVo细胞,在转染后的第24、48及72小时分别观察到逐渐增强的绿色荧光蛋白的表达.分别应用RT-PCR和Western blot法检测Bag-1在LoVo细胞中的表达,可见Bag-1在LoVo细胞中的mRNA和蛋白表达水平均较高.在Bag-1有效shRNA干扰序列筛选实验中,综合定量PCR及Western blot内源筛靶结果,筛选出Bag-1-homo-825作为最佳干扰靶序列.结论 成功构建针对小鼠Bag-1基因的特异性shRNA质粒,获得稳定转染的结肠癌Lovo细胞株,结果证明所构建的pGPH1/GFP/Neo-Bag-1-homo-825作为最佳干扰靶序列,显著抑制Lovo细胞Bag-1 mRNA和蛋白的表达.
目的 構建Bag-1短髮卡RNA(shRNA)榦擾載體併通過內源性篩靶實驗檢測Bag-1基因mRNA和蛋白的錶達,篩選齣最佳的榦擾靶序列.方法 應用榦擾技術構建Bag-1 shRNA榦擾載體,在細胞轉染預實驗中將構建好的帶有綠色熒光蛋白的質粒轉染Lovo細胞,通過觀察綠色熒光蛋白來判斷轉染的效率,分彆應用逆轉錄-聚閤酶鏈反應(RT-PCR)和Western blot法檢測Bag-1在LoVo細胞中的錶達.在shRNA榦擾序列篩選實驗中,通過定量PCR和Western blot法來檢測構建的榦擾載體在LoVo細胞中的錶達.結果 重組質粒分彆經雙酶切分析,瓊脂糖凝膠電泳鑒定可見清晰地切齣與Bag-1及pGPHl/GFP/Neo載體大小相符的片段併與DNA Marker相符.用帶綠色熒光基因的pGPH1/GFP/Neo-shNC質粒轉染LoVo細胞,在轉染後的第24、48及72小時分彆觀察到逐漸增彊的綠色熒光蛋白的錶達.分彆應用RT-PCR和Western blot法檢測Bag-1在LoVo細胞中的錶達,可見Bag-1在LoVo細胞中的mRNA和蛋白錶達水平均較高.在Bag-1有效shRNA榦擾序列篩選實驗中,綜閤定量PCR及Western blot內源篩靶結果,篩選齣Bag-1-homo-825作為最佳榦擾靶序列.結論 成功構建針對小鼠Bag-1基因的特異性shRNA質粒,穫得穩定轉染的結腸癌Lovo細胞株,結果證明所構建的pGPH1/GFP/Neo-Bag-1-homo-825作為最佳榦擾靶序列,顯著抑製Lovo細胞Bag-1 mRNA和蛋白的錶達.
목적 구건Bag-1단발잡RNA(shRNA)간우재체병통과내원성사파실험검측Bag-1기인mRNA화단백적표체,사선출최가적간우파서렬.방법 응용간우기술구건Bag-1 shRNA간우재체,재세포전염예실험중장구건호적대유록색형광단백적질립전염Lovo세포,통과관찰록색형광단백래판단전염적효솔,분별응용역전록-취합매련반응(RT-PCR)화Western blot법검측Bag-1재LoVo세포중적표체.재shRNA간우서렬사선실험중,통과정량PCR화Western blot법래검측구건적간우재체재LoVo세포중적표체.결과 중조질립분별경쌍매절분석,경지당응효전영감정가견청석지절출여Bag-1급pGPHl/GFP/Neo재체대소상부적편단병여DNA Marker상부.용대록색형광기인적pGPH1/GFP/Neo-shNC질립전염LoVo세포,재전염후적제24、48급72소시분별관찰도축점증강적록색형광단백적표체.분별응용RT-PCR화Western blot법검측Bag-1재LoVo세포중적표체,가견Bag-1재LoVo세포중적mRNA화단백표체수평균교고.재Bag-1유효shRNA간우서렬사선실험중,종합정량PCR급Western blot내원사파결과,사선출Bag-1-homo-825작위최가간우파서렬.결론 성공구건침대소서Bag-1기인적특이성shRNA질립,획득은정전염적결장암Lovo세포주,결과증명소구건적pGPH1/GFP/Neo-Bag-1-homo-825작위최가간우파서렬,현저억제Lovo세포Bag-1 mRNA화단백적표체.
Objective To construct the short hairpin RNA (shRNA) plasmids carrying Bag-1 gene of human and test the expression of Bag-1 mRNA and protein.Methods Recombinant plasmids containing green fluorescent protein reporter genes were constructed using gene cloning methods.The shRNA plasmids for Bag-1 gene were constructed by RNA interference technology.Fluorescent plasmid-transfected target cells were used in the cell transfection experiments,and the transfection efficacy of plasmids was analyzed by observing the fluorescence amount.Three synthetized shRNAs were transfected into target screening cells,and reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to identify the difference of target gene transfection and translation level in cells.Results The specific shRNA plasmid for Bag-1 gene was successfully recombinanted and stably transfected colon cancer LoVo cell lines were obtained.The constructed shRNA plasmids significantly inhibited the expression of Bag-1 mRNA and protein of Lovo cells,and could maintain the effect for a long term.Conclusion pGPH1/GFP/ Neo-Bag-1-homo-825 was screened as the optimum sequence of interference,and can significantly inhibit the expression of Bag-1 mRNA and protein.
