本文選題：人膀胱移行上皮癌 + Robo　； 參考：《廣東藥學(xué)院》2014年碩士論文

【摘要】：背景： 膀胱癌是泌尿外科常見的惡性腫瘤，其病因目前仍不清楚。治療上除依賴早期診斷、手術(shù)切除外并無特殊有效的方法。神經(jīng)導(dǎo)向因子Slit家族最先發(fā)現(xiàn)在調(diào)節(jié)神經(jīng)元前體細胞定向遷移和神經(jīng)元軸突的定向生長中具有重要作用[1]。近期研究發(fā)現(xiàn)Slit/Robo信號通路在調(diào)節(jié)腫瘤細胞遷移、腫瘤血管生成等方面發(fā)揮作用，還有少量研究發(fā)現(xiàn)Robo在乳腺癌、肝癌等腫瘤細胞中有表達[2,3]。已有的這些研究表明Slit/Robo信號很可能是腫瘤發(fā)生和轉(zhuǎn)移的一條重要信號通路。 為進一步研究Robo信號蛋白在膀胱癌病理發(fā)生中的作用并探討治療的方法。我們擬在體外培養(yǎng)人膀胱移行上皮癌T24細胞，并檢測Robo蛋白在人膀胱癌移行上皮細胞T24中的表達。若該蛋白在T24細胞中表達，擬用RNA干擾技術(shù)沉默該基因并觀察腫瘤細胞的生長情況。嘗試探索膀胱癌的病理機制及實驗性的治療方法。 研究方法： 1、體外培養(yǎng)人膀胱移行上皮癌T24細胞，并用免疫組織化學(xué)染色法檢測是否有Robo蛋白的表達。如果有表達，表達何種亞型？ 2、用免疫熒光和蛋白質(zhì)印跡法進一步證實免疫組織化學(xué)的結(jié)果 3、利用RNA干擾技術(shù)，，尋找表達基因的沉默位點，構(gòu)建合理的表達載體，轉(zhuǎn)染膀胱移行上皮癌T24細胞，免疫熒光化學(xué)觀察細胞轉(zhuǎn)染結(jié)果，western blot來檢測該表達基因沉默后蛋白表達的變化。 4、 MTT法檢測該表達基因沉默后細胞的存活率的變化。 研究結(jié)果： 1、免疫組化和免疫熒光檢測顯示在體外培養(yǎng)的人膀胱移行上皮癌細胞T24中有Robo陽性細胞檢出。分類抗體染色顯示表達亞型為Robo1和Robo4，而正常膀胱移行上皮細胞中無表達。 2、 Western blot檢測顯示在人膀胱移行上皮癌細胞T24中有Robo1和Robo4蛋白表達。 3、采用RNA干擾技術(shù)能有效沉默T24中Robo1和Robo4的表達，尤其是Robo1的表達幾乎受到全部抑制（P＜0.01）。 4、 MTT法檢測顯示Robo1干擾組細胞的存活率明顯下降，與對照組及Robo4組相比差異有顯著性（P＜0.05）。 結(jié)論： 1、在人膀胱移行上皮癌細胞T24中存在Robo1和Robo4兩種蛋白受體。 2、利用RNA干擾技術(shù)干擾Robo1和Robo4后，Robo1的表達明顯下降且腫瘤細胞的存活率明顯下降。提示Robo基因可能在膀胱癌的生長中起重要作用，有可能成為膀胱癌腫瘤基因治療的新靶點。
[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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.14

【參考文獻】

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1 刁鑫偉;王欽文;謝啟超;陳正堂;葉明福;湯金梁;;逆轉(zhuǎn)錄病毒介導(dǎo)重組CXCR4對前列腺癌細胞侵襲力的影響[J];現(xiàn)代腫瘤醫(yī)學(xué);2010年02期

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