摘要
目的:研究利用RNA干扰抑制Survivin基因的表达对人乳腺癌SKBr-3细胞放射敏感性的影响。方法:构建针对Sur-vivin基因的RNA干扰真核表达载体pSU-PER-S1并转染SKBr-3细胞,分别用RT-PCR和蛋白质印迹法检测Survivin mR-NA和蛋白表达水平的变化;流式细胞仪检测细胞周期;平板克隆形成实验计算细胞存活率,用单击多靶数学模型进行曲线拟合做图,求曲线参数D0、Dq和N值,比较细胞放射敏感性变化。结果:RT-PCR和蛋白质印迹法检测结果表明,pSUPER-S1转染后的SKBr-3细胞中Survivin mRNA和蛋白水平显著下降;流式细胞仪检测结果显示,转染后的SKBr-3细胞G1期阻滞明显〔(74.03±8.91)%〕,高于对照组〔(44.21±12.03)%〕,S期细胞数减少〔(17.11±4.96)%〕,低于对照组〔(43.12±8.64)%〕,P<0.05;经不同剂量γ射线照射后的细胞存活曲线拟合作图后发现,转染后的细胞D0、N和Dq值均下降(D0=1.29,N=4.9,Dq=2.6),与对照组相比(D0=1.56,N=14.85,Dq=5.21)差异有统计学意义(P<0.05),提示细胞放射增敏感性增加。结论:利用RNA干扰技术抑制Survivin基因在乳腺癌SKBr-3细胞中的表达可以增加其对放疗的敏感性。
OBJECTIVE： To investigate the effect of inhibiting Survivin expression by RNA interference on the radiosensitivity of SK- Br-3 breast cancer cells. METHODS： A recombinant eukaryotic expression vector pSUPER-S1 carrying the specific siRNA of Survivin gene was introduced by lipofectamine mediated gene transfection into cultured SKBr-3 cells. Stably transfected cell clones were collected and analyzed by RT-PCR to determine the level of Survivin mRNA. The protein level of Survivin was detected by Western blot. The cell cycle phases were determined by flow cytometry （FCM）. The radiosensitivity of the ceils was examined by colony forming assay, and the values of D0, Dq and N were estimated. RESULTS： In the experiment group, both the mRNA and protein expression levels of Survivin gene in the transfected SKBr-3 cells were reduced significantly, as detected by RT-PCR and Western blot. FCM analysis revealed that most of the transfected SKBr-3 cells were blocked in G1 phase of cell cycle [（74. 03±8. 91 ）%], which were more than those of the control group [（44. 21±12. 03）%], P〈0. 05. The transfection rate in S phase [（17. 11±4.96）%] was less than that in the control group [（43. 12±8. 64）%], P〈0. 05. After the irradiation of different doses of γ-ray, the values of D0, N and Dq were ob tained through the cell survival curves. It showed that the values of transfected SKBr-3 cells （D0= 1.29,N 4. 9,Dq=2. 6） were lower than those in control group（D0= 1.56, N= 14. 85, Dq= 5.21） significantly （P〈0. 05）, which demonstrated that the radiosensitivity of these cells was enhanced. CONCLUSION： RNAi can markedly inhibit the expression of Survivin gene and subsequently increase the radiosensitivity of cancer cells so as to improve the therapeutic effect.
出处
《中华肿瘤防治杂志》
CAS
2008年第20期1528-1531,共4页
Chinese Journal of Cancer Prevention and Treatment
基金
国家自然科学基金(30500433)
河北省科技支撑计划项目(072761677)
作者简介
【通讯作者简介】李庆霞，女，河北临漳人，博士，副主任医师，副教授，主要从事肿瘤内科的临床与基础研究工作。Tel：86-311—85989933 E-mail：lqx73@163．com
