下调Notch-1基因表达抑制胶质母细胞瘤的增殖活性研究

Down- regulation of Notch- 1 gene expression inhibits growth of TJ- 905 glioblastoma

目的 应用siRNA下调胶质母细胞瘤TJ-905细胞株Notch-1基因抑制肿瘤增殖活性.方法 实验设以Notch-1为基因靶点的siRNA1、siRNA2、siRNA3转染组、阴性对照组和空白对照组,采用OligofectamineTM2000二次转染方法将 siRNA转移到细胞内.实时荧光定量PCR检测siRNA对Notch-1基因表达的抑制作用,筛选出最优siRNA序列用于后续研究;四唑盐（MTT）比色法检测细胞增殖活性;使用Notch-1 siRNA或阴性siRNA转染的TJ-905细胞悬液注射裸鼠腋下,观察移植瘤生长情况,比较肿瘤体积,观察3周后处死动物,切片行HE染色鉴定.结果 （Ⅰ）siRNA明显抑制Notch-1 mRNA的表达水平,空白对照组、阴性对照组、siRNA1、2、3转染组Notch-1 mRNA 表达率分别为1.00±0.07、1.04±0.05、0.11±0.02、0.12±0.01、0.77±0.03,siRNA1为最优序列.（2）siRNA1转染组细胞增殖活性显著降低.（3）接种Notch-1 siRNA1转染的TJ-905细胞裸鼠肿瘤生长速度明显低于接种阴性转染的TJ-905细胞裸鼠,裸鼠处死后观察肿瘤体积：Notch-1 siRNA1转染组为（748.3±154.3）mm3,阴性转染组为（1739.2±249.7）mm3,病理切片HE染色证明为多形性胶质母细胞瘤.结论 化学合成的靶向Notch-1基因的siRNA可以显著地下调Notch-1基因表达水平,抑制肿瘤增殖活性.为进一步进行胶质瘤的基因治疗研究奠定基础.

Objective To study the inhibitory effect of siRNA on glioblastoma （GBM） Notch-1 gene expression in addition to the growth of TJ- 905 glioblastoma.Methods Three Small interference RNAs （siRNAs） targeting Notch- 1 gene named siRNA1,siRNA2,siRNA3 were synthesized chemically in vitro with gene bank BLAST.TJ-905 cells were transfectedtwice with the siRNA by using OligofectamineTM2000.The nontransfected cells and nonspecific siRNAs transfected cells were taken as blank and nonsense controls.Down - regulation of Notch- 1 was demonstrated by real- time RT- PCR,according to the result of QRT- PCR we therefore selected the most effective siRNA for further study.Cell proliferation was measured by MTT analysis.Male Balb/C nude mice were injected subcutaneously with Notch- 1 siRNA- or nonsense siRNA- transfected TJ-905 cells,tumor sizes were measured every 4 days.HE stain was used to determine the property of tumor.Resuts（1） The expression of Notch- 1 mRNA in blank,nonsense controls,siRNA1,2,3 groups were 1.00±0.07,1.04±0.05,0.11 ±0.02,0.12 ±0.01,0.77 ±0.03 by real-time RT- PCR scan analysis,the most effective siRNA was siRNA1.（2） An inhibitory proliferation and growth can be induced in siRNA1 transfected group.（3）Nude mice xenografted with cell suspensions from the nonsense siRNA group developed tumors with a significantly increased volume （from the 18th days） as compared to mice that received the Notch- 1 siRNA1- treated cells.The final tumor volume were less in nude mice injected with Notch- 1 siRNA （748.3 ±154.3 ）mm3 compared to nonsense si RNA injection （1739.2 ± 249.7 ）mm3,HE stain demonstrate that tumor was multiformity glioblastoma.Conclusion The chemically synthesized siRNA targeted Notch- 1 gene could down -regulate the expression of Notch- 1.In addition,an inhibitory proliferation and growth were induced when compared with the control cells in vitro and in vivo.It was suggested that the suppression of Notch- 1 expression and the inhibition of growth provide a new way to glioma gene therapy.