Serpin B3对大鼠乳腺癌Walker256细胞骨转移的影响

摘要

目的 通过慢病毒干扰技术丝氨酸蛋白酶抑制剂B3(serine proteinase inhibitor B3,Serpin B3)在Walker256细胞株中的表达,建立乳腺癌骨转移瘤模型,探讨Serpin B3对大鼠乳腺癌Walker256细胞骨转移的影响.方法 构建靶向干扰Serpin B3(RNAi)慢病毒,感染乳腺癌Walker256细胞,以干扰Scramble序列作为对照,Real-time PCR和Western blot检测Serpin B3干扰效率.将32只SPF级雌性SD(sprague-dawley)大鼠随机均分为四组:阴性对照组、阳性对照组、骨癌痛阴性病毒对照组(LV-Scrl组)、骨癌痛干扰病毒组(LV-Serpin B3 shRNA组),各组于右腿胫骨平台分别注射20μl灭菌PBS、Walker256细胞、转染Scrl的Walker256细胞、转染RNAi的Walker256细胞.模型构建成功后21 d,Real-time PCR和Western blot检测各组大鼠转移瘤中Serpin B3 mRNA、蛋白表达含量;取各组大鼠患侧胫骨组织行甲苯胺蓝染色,观察肿瘤组织病理变化.结果 阳性对照组Serpin B3在乳腺癌细胞中高表达;与阳性对照组相比,LV-Serpin B3 shRNA在大鼠Walker 256细胞高效率表达,导致在骨转移瘤组织中Serpin B3 mRNA、蛋白表达明显降低(均P<0.05),且患侧骨组织破坏程度减轻.结论 Serpin B3在乳腺癌转移瘤组织中高表达,干扰Serpin B3表达能减弱乳腺癌Walker256细胞的骨转移倾向.%Objective To investigate the effects of serine proteinase inhibitor B3 (Serpin B3) on the bone metastasis of rat breast cancer cell line Walker256. Methods Lentiviral vector targeting Serpin B3 (RNAi) was constructed to infect Walker256 cells, and lentiviral vector with scramble sequence was used as control. The quantitative real-time PCR and Western blotting were used to detect the expression of Serpin B 3. The 32 female rats were divided into four groups:negative control group, positive control group, LV-Scrl group, LV-serpin b3 shRNAi group, which were respectively injected with 20 μL sterile PBS, Walker256 cells, Walker256 cells transfected with Scrl, Walker256 cells transfected with RNAi in the right tibia platform of rats. On the day 21, quantitative real-time PCR and Western blotting were used to test the mRNA and protein expression of Serpin B3 in metastatic bone tumor;and the right tibia tissues were separated for observation of the pathological changes by toluidine blue staining. Results The expression of Serpin B3 in the breast cancer tissues was high in positive control group. As compared with positive control group, Lentiviral vector targeting Serpin B3 could be expressed effectively in rats Walker256 cells, resulting in the significant decrease in the mRNA and protein expression of Serpin B3 (P<0.05), and the less destruction of bone tissuein LV-Serpin B3 shRNAi group. Conclusion Serpin B3 was highly expressed in breast carcinoma, and interfering Serpin B3 expression can decrease the tendency of breast cancer bone metastases.