摘要
目的 :研究siRNA(smallinterferenceRNA)对bcl- 2基因表达的抑制作用。方法 :依据Ambion公司在网上提供的设计软件 ,设计合成以bcl- 2基因为靶标的siRNA ,通过脂质体将合成的siRNA转入U2 5 1细胞株 ,以未转染细胞以及bcl- 2的反义药物G3139为对照 ,经MTT法检测siRNA对细胞生长的抑制以及流式细胞仪检测细胞周期的改变 ,用RT -PCR和免疫组织化学法检测siRNA对bcl- 2表达的抑制作用。结果 :MTT显示各时点细胞生长以及存活率 ,在siRNA 2、3、5、6与siRNA 1、4组以及对照组和脂质体组间均有显著差异 (P <0 0 5 )。siRNA 1、4组与对照组和脂质体组也有差异 (P <0 0 5 )。siRNA 1- 6组与反义组在 2 4和 4 8h均有显著差异 (P <0 0 5 ) ,在 72h无显著差异 (P >0 0 5 )。RT -PCR以及免疫组织化学法显示 ,siRNA 2、3、5、6组bcl- 2基因表达明显低于对照组、脂质体组、反义组以及siRNA 1、4组 (P <0 0 5 )。流式细胞仪检测细胞周期结果显示 ,siRNA 1- 6组以及反义组细胞阻滞于S期。结论 :体外转录合成的siRNA可抑制U2 5 1细胞bcl- 2基因的表达 ,效率可达 5 0 %以上。
AIM: To evaluate the effectiveness of small interference RNA on inhibiting bcl-2 gene expression. METHODS: With Ambion's software and kit, we designed and synthesized siRNA targeted bcl-2, which were transfected into astrocytoma cell line U251 with lipofectamine. The non-transfected cells and treatment with antisense drug G-3139 were taken as controls. MTT was used to detect the inhibitory rate of cell growth. Flow cytometric method was used to detect the change in cycle of the cells. The inhibitory effect of siRNA on mRNA level was detected by RT-PCR and on protein level was by immunohistochemical method. RESULTS: For the living rate of cell, siRNA 2, 3, 5, 6 groups were significantly lower than siRNA 1, 4 groups, lipofectamine and control group at 24, 48, and 96 h. siRNA 1-6 groups only had statistic difference with antisense group at 24, 48 h. As for PCR and immunohistochemical method, the expression of bcl-2 on siRNA 2, 3, 5, 6 groups were significant lower than other groups. The results of flow cytomytric method showed the cells transfected with siRNA 1-6 and antisense were blocked at S stage. CONCLUSION: siRNA inhibited bcl-2 gene expression more than 50%.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2005年第3期489-493,共5页
Chinese Journal of Pathophysiology
基金
广东省自然科学重点基金资助项目 (No .0 2 1195 )
