CAJ | 학술논문

目的：针对雌激素受体（ ER）-α36基因的特异性靶序列，构建并鉴定靶向ER-α36基因RNAi慢病毒载体，研究ER-α36沉默后对胃癌细胞增殖的影响。方法：筛选确定的ER-α36基因RNAi有效靶序列，合成靶序列的Oligo DNA，与慢病毒载体（GV307）连接，测序鉴定。转染293T细胞，包装产生慢病毒，感染胃癌SGC7901细胞株。荧光显微镜下观察SGC7901感染后荧光表达情况，real-time PCR和Western blotting方法检测ER-α36的表达变化。用1×10－10 mol／L的17β-雌二醇处理沉默ER-α36的SGC7901细胞，用细胞计数法观察细胞增殖能力的变化及检测相关下游信号通路分子Src、ERK1／2、cyclin D1表达的变化。结果：阳性克隆PCR及测序证明成功构建慢病毒载体LV-ER-α36-RNAi，倒置显微镜下观察LV-ER-α36-RNAi慢病毒载体感染率达80％以上。 Real-time PCR和Western blotting方法证实四环素（ TeT）诱导下LV-ER-α36-RNAi明显抑制SGC7901细胞内ER-α36 mRNA和蛋白质的表达。与对照组相比，沉默ER-α36的SGC7901细胞增殖能力减弱，Src、ERK1／2、cyclin D1蛋白表达明显降低，Src蛋白活化能力减弱（ P＜0.05）。结论：我们构建的TeT诱导靶向ER-α36的vshRNA慢病毒载体LV-ER-α36-RNAi，可明显沉默ER-α36的表达，为研究ER-α36蛋白的作用机理提供实验依据，ER-α36与胃癌细胞等肿瘤细胞的增殖有关。
목적：침대자격소수체（ ER）-α36기인적특이성파서렬，구건병감정파향ER-α36기인RNAi만병독재체，연구ER-α36침묵후대위암세포증식적영향。방법：사선학정적ER-α36기인RNAi유효파서렬，합성파서렬적Oligo DNA，여만병독재체（GV307）련접，측서감정。전염293T세포，포장산생만병독，감염위암SGC7901세포주。형광현미경하관찰SGC7901감염후형광표체정황，real-time PCR화Western blotting방법검측ER-α36적표체변화。용1×10－10 mol／L적17β-자이순처리침묵ER-α36적SGC7901세포，용세포계수법관찰세포증식능력적변화급검측상관하유신호통로분자Src、ERK1／2、cyclin D1표체적변화。결과：양성극륭PCR급측서증명성공구건만병독재체LV-ER-α36-RNAi，도치현미경하관찰LV-ER-α36-RNAi만병독재체감염솔체80％이상。 Real-time PCR화Western blotting방법증실사배소（ TeT）유도하LV-ER-α36-RNAi명현억제SGC7901세포내ER-α36 mRNA화단백질적표체。여대조조상비，침묵ER-α36적SGC7901세포증식능력감약，Src、ERK1／2、cyclin D1단백표체명현강저，Src단백활화능력감약（ P＜0.05）。결론：아문구건적TeT유도파향ER-α36적vshRNA만병독재체LV-ER-α36-RNAi，가명현침묵ER-α36적표체，위연구ER-α36단백적작용궤리제공실험의거，ER-α36여위암세포등종류세포적증식유관。
[ ABSTRACT] AIM:To construct a lentiviral vector for stable delivery of the ER-α36 gene and to detect its effect on SGC7901 cell growth.METHODS: The efficient RNAi targeting sequences identified for the ER-α36 gene were screened.The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA.Then it was digested by Xho I and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector.PCR was used to screen the positive clones and sequence.The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line.Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect.17β-estrodial at concentration of 1 ×10 -10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D1 were determined.RESULTS: DNA sequencing analysis confirmed the identity of recombinant shRNA expression vectors.Immunofluorescence assay demonstrated that transfection efficiency was above 80%.Transfection of LV-ER-α36-RNAi significantly knocked down the expression of ER-α36 at mR-NA and protein levels with tetracycline ( TeT) simulating as revealed by real-time PCR and Western blotting.Compared with control group, the growth of the recombinant cell line declined and the expression of Src, ERK1/2 and cyclin D1 and the activation of Src decreased (P<0.05).CONCLUSION: Lentiviral vectors that silence ER-α36 expression are con-structed successfully and can be used to study the role of ER-α36 in gastric cancer.The ER-α36 is related with many kinds of cancer cell growth, including gastric cancer cells.