CAJ | 학술논문

目的利用RNA干扰(RNAi)技术,以survivin为靶基因,设计针对survivin的siRNA,通过Hiperfect介导转染K562细胞,研究survivin基因在白血病发生发展中的作用,为以survivin为靶点的白血病治疗奠定基础.方法体外化学合成抑制survivin基因的siRNA片段,通过Hiperfect转染到K562细胞内,以无关siRNA、Hiperfect和未转染的细胞为对照,采用SYBR Green荧光染料Ⅰ的实时荧光定量RT-PCR方法检测siRNA的抑制效果,利用流式细胞仪Annexin Ⅴ-FITC/PI法检验细胞凋亡率,用四氮唑盐(MTT)法观察转染后48 h,72 h对细胞增生的影响.结果 siRNA转染K562细胞后,SYBR Green荧光染料Ⅰ的实时荧光定量RT-PCR结果显示survivin mRNA的表达量48 h、72 h分别比空白组下降了85.21%、94.35%,细胞生长受抑制,增殖能力减弱,其增殖抑制率于转染后48 h、72 h分别为45.02%和50.88%,细胞的凋亡率升高,分别为12.28%、21.55%.结论靶向survivin的siRNA能特异性下调目的基因的表达、并能在体外抑制白血病细胞株的生长.survivin将成为白血病基因治疗的一个新靶点.
목적 이용RNA간우(RNAi)기술,이survivin위파기인,설계침대survivin적siRNA,통과Hiperfect개도전염K562세포,연구survivin기인재백혈병발생발전중적작용,위이survivin위파점적백혈병치료전정기출.방법 체외화학합성억제survivin기인적siRNA편단,통과Hiperfect전염도K562세포내,이무관siRNA、Hiperfect화미전염적세포위대조,채용SYBR Green형광염료Ⅰ적실시형광정량RT-PCR방법검측siRNA적억제효과,이용류식세포의Annexin Ⅴ-FITC/PI법검험세포조망솔,용사담서염(MTT)법관찰전염후48 h,72 h대세포증생적영향.결과 siRNA전염K562세포후,SYBR Green형광염료Ⅰ적실시형광정량RT-PCR결과현시survivin mRNA적표체량48 h、72 h분별비공백조하강료85.21%、94.35%,세포생장수억제,증식능력감약,기증식억제솔우전염후48 h、72 h분별위45.02%화50.88%,세포적조망솔승고,분별위12.28%、21.55%.결론 파향survivin적siRNA능특이성하조목적기인적표체、병능재체외억제백혈병세포주적생장.survivin장성위백혈병기인치료적일개신파점.
Objective To evaluate the impact of specific siRNA on survivin gene in transfected leukemia cells. Method The small interfering RNA(siRNA) targeted survivin mRNA was synthesized in vitro and was transfected into K562 cell by Hiperfect into human leukemia cell line K562, which has high survivin expression level The level of survivin mRNA expression was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR) with SYBR GREEN Ⅰ . The apoptosis index of cytotrophoblasts were dertermined and analysized by FCM ( Annexin Ⅴ-FITC/PI staining methods). The cell proliferation was examined by MTT at 48 h and 72 h after transfection. Result The level of mRNA expression was significantly inhibited by the siRNA 48 h and 72 h after transfection, the suppression rate of survivin mRNA separately reached 85.21%, 94.35% mensurated by quantitative RT-PCR with SYBR GREEN Ⅰ , cell proliferation was inhibited significantly by 45.02% and 50.88%, respectively, the apoptotic rate detected by Annexin Ⅴ-FITC assay reached 12.28% and 21.55%, respectively. Conclusion The chemosynthesized siRNA targeting survivin could significantly down-regulate survivin mRNA. Survivin siRNA was able to inhibit the proliferation of leukemia cell line K562. Survivin may become a new target for leukemia gene therapy.