双基因shRNA干扰乙酰肝素酶在卵巢癌细胞中表达的实验研究

Inhibition of heparanase gene expression by double shRNA expressing vector in ovarian carcinoma cells

目的：采用编码2条短发卡式RNA(shRNA)的抗乙酰肝素酶(heparanase,Hpa)特异性载体干扰卵巢癌细胞中Hpa的表达,观察其对肿瘤生长转移的抑制作用。方法：以脂质体介导的方法将自行设计和构建的抗Hpa短发卡式RNA (Hpa-shRNA)基因真核表达载体稳定转染人卵巢癌细胞SKOV3；流武细胞仪鉴定转染效率,免疫组化和实时定量RT-PCR检测转染前后卵巢癌细胞中Hpa蛋白和mRNA表达,Boyden小室观测细胞增殖和侵袭能力的变化,透射电镜观察卵巢癌细胞超微结构改变。结果：Hpa mRNA表达在SKOV_3-Hpa(转染Hpa)明显升高(P＜0．01),SKOV3-Hpa-shRNA(转染Hpa- sh RNA)显著降低(P＜0．01),SKOV3-DZ-shRNA(转染非特异性sh RNA片段)和SKOV3无显著性差异(P＞0．05)。电镜下,SKOV3-Hpa组Hpa蛋白表达最为明显,SKOV3-Hpa-shRNA Hpa蛋白则最低,各组间比较差别有显著性意义(P＜0．01)。SKOV3-Hpa细胞器发达,细胞表面绒毛丰富,细胞间连接稀少。SKOV3-Hpa-shRNA以变形为主,细胞表面绒毛少,细胞器不发达,部分见线粒体皱缩,肿胀和空泡样改变。SKOV3-Hpa、SKOV3-DZ-shRNA和SKOV3-Hpa-shRNA 3种细胞穿过人工基底膜的细胞数分别为82．3±9．16,36．1±8．73和18．6±10．5,各组间比较有显著性差异(P＜0．01)。结论：双基因Hpa特异性shRNA可显著抑制卵巢癌细胞Hpa表达,降低细胞侵袭能力。

Objective：To explore whether Hpa-targeted double short hairpin RNA （shRNA） could interfere with the expression of heparanase（Hpa）gene in ovarian carcinoma cells and inhibit the growth and metastasis of tumor cells. Methods： Self-designed and constructed Hpa-targeted shRNA eukaryotic expression vector was introduced into ovarian cancer SKOV3 cells via lipofectin mediation. The positive clones were screened by gengticin （G418）. The transfection efficiency was detected by flow cytometry. The expression of Hpa mRNA and protein in SKOV3 cells was confirmed by FQ Real-time RT-PCR and immunohistochemical staining before or after transfection. The proliferation and invasion ability of SKOV3 cells was assesaed by Boyden Chamber assay. Micro-morphologic changes of SKOV3 cells were observed under electron transmission microscope. Results：The expression of Hpa mRNA significantly increased in SKOV3-Hpa group and significantly decreased in SKOV3-Hpa-shRNA group （P〈0.01）. The difference between SKOV3-DZ-shRNA group and SKOV3 group was not significantly different （P〈0. 05）. Electron transmission microscopy showed that the expression of Hpa protein was highest in SKOV3-Hpa group and lowest in SKOV3-Hpa-shRNA group. The difference between each group was significant （P〈0.01）. SKOV3-Hpa cells had abundant cellular organelles, enriched villa on the surface, and sparse cell-cell junctions. Most SKOV3-Hpa-shRNA cells degenerated. The villa and cellular organelle were reduced. Some cells showed condensed, swollen, or vacuolated mitochondria. The Boyden Chamber assay showed that migrated cells was 82.3±9.16, 36.1±8. 73, and 18.6±10. 5 in SKOV3-Hpa, SKOV3-DZ-shRNA, and SKOV3-Hpa-shRNA group, respectively. The difference between each group was significant （P〈0. 01）. Conclusions： Hpa-targeted shRNA specifically interfered with the expression of Hpa mRNA in ovarian carcinoma cells and significantly inhibited the invasion ability of cells.