目的: 研究siRNA对卵巢癌细胞株SKOV3中乙酰肝素酶(heparanase,HPA)基因表达的抑制作用。方法: 设计合成两对HPA编码基因的反向重复序列，中间间隔9个核苷酸序列，通过定向克隆至载体Pgenesil-1，构建siRNA真核表达载体，经稳定转染SKOV3细胞后应用RT-PCR、real-time PCR及免疫组化技术检测卵巢癌细胞中HPA基因mRNA及蛋白表达水平的抑制情况。结果: 测序证实成功地构建了编码2条shRNA的siRNA真核表达载体。通过RT-PCR、real-time PCR及免疫组化技术检测转染siRNA的SKOV3细胞，与对照组相比，HPA mRNA表达水平和蛋白表达水平明显降低。结论:构建的PGenesil-1(+)-HPA重组质粒能有效的抑制HPA基因在卵巢癌细胞株SKOV3中的表达，为研究HPA基因在肿瘤细胞中的调节途径和肿瘤的基因治疗提供了新的方法。

Objective: To study the inhibitory effect of siRNA on Heparanase (HPA) expression in SKOV3 cells. Methods:Two pairs of 21 bp reverse repeated sequence targeting HPA RNA (spaced by 9 bp nucleotide) were synthesized and were cloned into plasmid Pgenesil-1 to construct recombinant plasmid PGenesil-1(+)-HPA expressing 2 hairpin siRNAs. The inhibition of HPA gene was detected by RT-PCR, real-time PCR and immunohistochemical staining after PGenesil-1(+)-HPA was stably transfected into SKOV3 cells. Results: The recombinant plasmid PGenesil-1(+)-HPA (expressing 2 hairpin siRNAs) was successfully constructed. RT-PCR, real-time PCR and immunohistochemical staining showed that there was a significant decrease in HPA mRNA and protein level in experimental group compared with those in control group. Conclusion: siRNA targeting HPA mRNA can specically suppress the expression of HPA gene in SKOV3 cells; RNA interference method provides a new way for studying the role of HPA and gene therapy of cancer.

中国博士后科学基金资助(2005037776)