CAJ | 학술논문

目的构建pcDNA3.1-AML1-ETO真核表达载体,并观察AML1-ETO融合蛋白在U937细胞内的表达并检测其对细胞增殖与分化的影响.方法以原核表达载体pCMV5-AML1-ETO为模板扩增AML1-ETO目的片段,将该基因重组于pcDNA3.1/V5-His-TOPO真核表达载体上,用脂质体转染技术将其导入 U937细胞,经G418筛选获得稳定转染的克隆,PCR检测AML1-ETO基因的整合,RT-PCR及 Western blot检测AML1-ETO mRNA和蛋白的表达,用锥虫蓝拒染人工计数法观察细胞增殖活性,流式细胞术检测髓系分化抗原表达的变化,瑞特染色法观察细胞形态改变.结果 pcDNA3.1-AML1-ETO经酶切鉴定及DNA测序证实序列完全正确,筛选出AML1-ETO基因高表达的亚克隆,证实AML1-ETO基因稳定转染到U937细胞中并得到表达;转染细胞增殖受抑(P<0.05),髓系分化抗原CD11b表达阳性率[(4.17±0.31)%]低于转染空载体和未转染对照组[(11.40±0.17)%、(11.03±0.15)%](P<0.001),形态呈低分化表现.转染细胞经TPA处理后,CD11b表达无明显变化(P>0.05).结论成功构建pcDNA3.1-AML1-ETO表达载体,并在真核细胞中得到了正确表达,AML1-ETO 基因能抑制U937细胞的增殖与分化,为进一步研究该基因致白血病的机制奠定了基础.
목적 구건pcDNA3.1-AML1-ETO진핵표체재체,병관찰AML1-ETO융합단백재U937세포내적표체병검측기대세포증식여분화적영향.방법 이원핵표체재체pCMV5-AML1-ETO위모판확증AML1-ETO목적 편단,장해기인중조우pcDNA3.1/V5-His-TOPO진핵표체재체상,용지질체전염기술장기도입 U937세포,경G418사선획득은정전염적극륭,PCR검측AML1-ETO기인적정합,RT-PCR급 Western blot검측AML1-ETO mRNA화단백적표체,용추충람거염인공계수법관찰세포증식활성,류식세포술검측수계분화항원표체적변화,서특염색법관찰세포형태개변.결과 pcDNA3.1-AML1-ETO경매절감정급DNA측서증실서렬완전정학,사선출AML1-ETO기인고표체적아극륭,증실AML1-ETO기인은정전염도U937세포중병득도표체;전염세포증식수억(P<0.05),수계분화항원CD11b표체양성솔[(4.17±0.31)%]저우전염공재체화미전염대조조[(11.40±0.17)%、(11.03±0.15)%](P<0.001),형태정저분화표현.전염세포경TPA처리후,CD11b표체무명현변화(P>0.05).결론 성공구건pcDNA3.1-AML1-ETO표체재체,병재진핵세포중득도료정학표체,AML1-ETO 기인능억제U937세포적증식여분화,위진일보연구해기인치백혈병적궤제전정료기출.
Objective To construct a pcDNA3. 1 -AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells. Methods AML1 -ETO gene was amplified by PCR from pCMV5-AML1-ETO and inserted into eukaryotic expression plasmid pcDNA3. 1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with G418 were isolated. The integration and the expression levels of AML1-ETO in transfectants were determined by PCR, RT-PCR and Western blot analysis respectively. Trypan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the morphologic changes of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry. Results The recombinant pcDNA3. 1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AML1-ETO subclone was established. AML1-ETO was expressed in U937 cells transfected with pcDNA3.1 -AML1-ETO. The growth of the monoclonal cells was inhibited evidently (P < 0. 05). The expression of CD11b in transfected group [(4. 17±0. 31)%] was lower than that in empty plasmid transfected group and non-transfected group [(11.40 ± 0. 17)% and (11.03 ± 0. 15) %] respectively (P < 0.001). Transfected cells displayed morphology of less differentiation. The expression level of CD11b was unchanged in transfected cells treated with TPA (P > 0. 05). Conclusion The eukaryotic expression vector for AML1ETO gene was successfully constructed and expressed in U937. AML1-ETO inhibits the proliferation and differentiation of transfected cells. It provides the basis for further study of mechanisms of AML1 -ETO in leukemogenesis.