CAJ | 학술논문

目的应用RNA干扰(RNAi)技术沉默视网膜母细胞瘤细胞株SO-Rb50中凋亡抑制因子Survivin的表达,观察其干扰效果及对细胞的影响.方法实验研究.体外化学法合成针对人Survivin基因的特异性小干扰RNA(siRNA)和对Survivin基因无沉默效果的阴性对照siRNA,利用转染试剂分别将siRNA转入SO-Rb50细胞株,通过半定量RT-PCR和Western blot检测其对SO-Rb50细胞中Survivin基因和蛋白表达的抑制作用,四甲基偶氮唑盐(MTT)法检测不同浓度siRNA对细胞增殖抑制率,流式细胞仪检测转染后细胞凋亡率和细胞周期改变,荧光显微镜下观察细胞凋亡形态.对多组间数据比较采用单因素方差分析,处理组与对照组比较采用Dunnett-t检验.结果 Survivin特异性siRNA转染细胞24 h后Survivin mRNA和蛋白表达水平明显下调,对照组中其表达则无明显改变.MTT法结果显示Survivin特异性siRNA浓度为35、70、100 nmol/L时均对SO-Rb50细胞增殖具有抑制作用(P＜0.05),且具有剂茸依赖性.流式细胞仪检测发现Survivin特异性siRNA组出现凋亡亚二倍体峰,细胞被阻滞于G0/G1期,较对照组增加了8.11%,G2/M期细胞比例减少了5.75%,S期细胞仅减少了2.26%.荧光显微镜下可见部分细胞出现染色质浓缩和凋亡小体等典型的凋亡形态学变化.结论针对Survivin的RNA干扰技术可有效地下调SO-Rb50细胞中Survivin基因表达,进而抑制SO-Rb50细胞增殖和诱导其凋亡,为视网膜母细胞瘤的基因治疗提供了重要的途径.
목적 응용RNA간우(RNAi)기술침묵시망막모세포류세포주SO-Rb50중조망억제인자Survivin적표체,관찰기간우효과급대세포적영향.방법 실험연구.체외화학법합성침대인Survivin기인적특이성소간우RNA(siRNA)화대Survivin기인무침묵효과적음성대조siRNA,이용전염시제분별장siRNA전입SO-Rb50세포주,통과반정량RT-PCR화Western blot검측기대SO-Rb50세포중Survivin기인화단백표체적억제작용,사갑기우담서염(MTT)법검측불동농도siRNA대세포증식억제솔,류식세포의검측전염후세포조망솔화세포주기개변,형광현미경하관찰세포조망형태.대다조간수거비교채용단인소방차분석,처리조여대조조비교채용Dunnett-t검험.결과 Survivin특이성siRNA전염세포24 h후Survivin mRNA화단백표체수평명현하조,대조조중기표체칙무명현개변.MTT법결과현시Survivin특이성siRNA농도위35、70、100 nmol/L시균대SO-Rb50세포증식구유억제작용(P＜0.05),차구유제용의뢰성.류식세포의검측발현Survivin특이성siRNA조출현조망아이배체봉,세포피조체우G0/G1기,교대조조증가료8.11%,G2/M기세포비례감소료5.75%,S기세포부감소료2.26%.형광현미경하가견부분세포출현염색질농축화조망소체등전형적조망형태학변화.결론 침대Survivin적RNA간우기술가유효지하조SO-Rb50세포중Survivin기인표체,진이억제SO-Rb50세포증식화유도기조망,위시망막모세포류적기인치료제공료중요적도경.
Objective To investigate cell proliferation and apeptosis status of human retinoblagtoma cell line SO-Rb50 after knockdown of Survivin gene by means of small interfering RNA(siRNA).Methods Survivin specific siRNA designed from the human gene sequence and nonsense siRNA(as a negative control)Waft transfected into SO-Rb50 cells.The inhibition of the expression of Survivin mRNA and protein levels were detected by reverse transcription-polymerage chain reaction(RT-PCR)and Western blot Proliferation inhibition rate of SO-Rb50 cells was analyzed by MTT assay.Apoptotic rate and cell cycle were analyzed by flow cytometry(FCM)and the apoptotic morphology was observed by fluorescent microscope.Results The expression of Survivin at both mRNA and protein level in specific siRNA group decreased significantly in comparison with untransfected group and nonsense siRNA group after 24 hours of transfection.The proliferation of SO-Rb50 was inhibited in the Survivin specific siRNA group at the concentrations of 35,70 and 100 nmoL/L(P＜0.05).Flow cytometry showed obvious apoptotic peak in Survivin specific group with an accumulation of cells in the G0/G1 phage and a decrease in G2/M phage and S phase.Typical apoptosis morphyology was also observed under fluorescent microscope.Conclusions Sunrivin specific siRNA could inhibit SO-Rb50 cell proliferation and induced apeptosis by knockdown of Survivin gene.Our data suggests that the use of Survivin-specific siRNA deserves further investigation as a novel approach to retinoblastoma therapy.