Robo蛋白在膀胱癌表达的检测及沉默Robo基因对肿瘤细胞影响的研究

发布时间：2018-06-24 16:39

本文选题：人膀胱移行上皮癌 + Robo　；参考：《广东药学院》2014年硕士论文

【摘要】：背景：膀胱癌是泌尿外科常见的恶性肿瘤，其病因目前仍不清楚。治疗上除依赖早期诊断、手术切除外并无特殊有效的方法。神经导向因子Slit家族最先发现在调节神经元前体细胞定向迁移和神经元轴突的定向生长中具有重要作用[1]。近期研究发现Slit/Robo信号通路在调节肿瘤细胞迁移、肿瘤血管生成等方面发挥作用，还有少量研究发现Robo在乳腺癌、肝癌等肿瘤细胞中有表达[2,3]。已有的这些研究表明Slit/Robo信号很可能是肿瘤发生和转移的一条重要信号通路。为进一步研究Robo信号蛋白在膀胱癌病理发生中的作用并探讨治疗的方法。我们拟在体外培养人膀胱移行上皮癌T24细胞，并检测Robo蛋白在人膀胱癌移行上皮细胞T24中的表达。若该蛋白在T24细胞中表达，拟用RNA干扰技术沉默该基因并观察肿瘤细胞的生长情况。尝试探索膀胱癌的病理机制及实验性的治疗方法。研究方法： 1、体外培养人膀胱移行上皮癌T24细胞，并用免疫组织化学染色法检测是否有Robo蛋白的表达。如果有表达，表达何种亚型？ 2、用免疫荧光和蛋白质印迹法进一步证实免疫组织化学的结果 3、利用RNA干扰技术，，寻找表达基因的沉默位点，构建合理的表达载体，转染膀胱移行上皮癌T24细胞，免疫荧光化学观察细胞转染结果，western blot来检测该表达基因沉默后蛋白表达的变化。 4、 MTT法检测该表达基因沉默后细胞的存活率的变化。研究结果： 1、免疫组化和免疫荧光检测显示在体外培养的人膀胱移行上皮癌细胞T24中有Robo阳性细胞检出。分类抗体染色显示表达亚型为Robo1和Robo4，而正常膀胱移行上皮细胞中无表达。 2、 Western blot检测显示在人膀胱移行上皮癌细胞T24中有Robo1和Robo4蛋白表达。 3、采用RNA干扰技术能有效沉默T24中Robo1和Robo4的表达，尤其是Robo1的表达几乎受到全部抑制（P＜0.01）。 4、 MTT法检测显示Robo1干扰组细胞的存活率明显下降，与对照组及Robo4组相比差异有显著性（P＜0.05）。结论： 1、在人膀胱移行上皮癌细胞T24中存在Robo1和Robo4两种蛋白受体。 2、利用RNA干扰技术干扰Robo1和Robo4后，Robo1的表达明显下降且肿瘤细胞的存活率明显下降。提示Robo基因可能在膀胱癌的生长中起重要作用，有可能成为膀胱癌肿瘤基因治疗的新靶点。
[Abstract]:Background: bladder cancer is a common malignant tumor in urology. There is no special and effective treatment except for early diagnosis and surgical resection. The slit family of neuronal guidance factors was first found to play an important role in regulating the directional migration of neuronal precursor cells and the directional growth of neuronal axons [1]. Recent studies have found that the Slit-Robo signaling pathway plays a role in regulating tumor cell migration and tumor angiogenesis, and a few studies have found that Robo is expressed in breast cancer, liver cancer and other tumor cells. These studies suggest that Slitr / Robo signal may be an important signal pathway for tumor development and metastasis. To further study the role of Robo signal protein in the pathogenesis of bladder cancer and explore the treatment methods. We intend to culture human bladder transitional cell carcinoma (T24) cells in vitro and detect the expression of Robo protein in human bladder transitional epithelial cells (T24). If the protein is expressed in T24 cells, RNA interference technique will be used to silence the gene and observe the growth of tumor cells. To explore the pathological mechanism and experimental treatment of bladder cancer. Methods: 1. Human bladder transitional cell carcinoma (T24) cells were cultured in vitro, and the expression of Robo protein was detected by immunohistochemical staining. If there is expression, which subtype is expressed? 2. Immunofluorescence and Western blotting are used to further confirm the result of immunohistochemistry. 3. RNA interference technique is used to find the silencing site of the expressed gene. A reasonable expression vector was constructed and transfected into T24 cells of bladder transitional cell carcinoma. The changes of protein expression after the silencing of the expressed gene were detected by western blot. 4. The survival rate of the cells after the silencing of the expressed gene was detected by MTT assay. Results: 1.Immunohistochemistry and immunofluorescence showed that Robo positive cells were detected in human bladder transitional epithelial cells (T24) cultured in vitro. The subtypes were Robo1 and Robo4, but not in normal bladder transitional epithelial cells. 2Western blot showed that Robo1 and Robo4 were expressed in T24 cells. The expression of Robo1 and Robo4 in T24 was effectively silenced by RNA interference. Especially, the expression of Robo1 was almost completely inhibited (P < 0.01). MTT assay showed that the survival rate of Robo1 interference group was significantly lower than that of control group and Robo4 group (P < 0.05). Conclusion: 1. There are two protein receptors Robo1 and Robo4 in human bladder transitional epithelial cell T24. 2. The expression of Robo1 and Robo4 were significantly decreased and the survival rate of tumor cells was decreased by RNA interference technique. It is suggested that Robo gene may play an important role in the growth of bladder cancer and may be a new target for gene therapy of bladder cancer.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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