【摘要】：背景 膀胱癌是我國泌尿外科臨床工作中最常見的惡性腫瘤中的一種。膀胱尿路上皮癌(bladder urothelial carcinoma, BUC)在膀胱癌中占的比例大約90%以上。目前膀胱癌的治療以手術(shù)為主,但近幾年新興的分子靶向治療,使我們看到了不通過手術(shù)治療腫瘤的希望。組織蛋白酶B (cathepsin B, CB)屬于木瓜蛋白家族,是溶酶體內(nèi)重要的一種巰基蛋白酶。近年來的研究發(fā)現(xiàn)組織蛋白酶B與腫瘤的侵襲和轉(zhuǎn)移等惡性表型相關(guān)。但尚無組織蛋白酶B與T24細(xì)胞遷移和侵襲相關(guān)性的體外研究。 目的 探討組織蛋白酶B在人膀胱尿路上皮癌T24細(xì)胞中的表達(dá)情況；通過組織蛋白酶B的特異性抑制劑CA-074降低組織蛋白酶B蛋白的表達(dá)及活性,觀察T24細(xì)胞的遷移和侵襲力的變化。 方法 (1)實驗準(zhǔn)備：購買并傳代培養(yǎng)實驗用T24細(xì)胞。將CA-074用二甲基亞砜(DMSO)溶解成需要濃度。 (2)實驗分組：正常培養(yǎng)的T24細(xì)胞為空白對照組；以不同終濃度的CA-074培養(yǎng)液培養(yǎng)的T24細(xì)胞分為4個實驗組,分別為0.5μ mol/L濃度組、1μ mol/L濃度組、5μ mol/L濃度組、10μ mol/L濃度組；以含有溶劑DMSO的培養(yǎng)基培養(yǎng)的T24細(xì)胞作為實驗對照組。分別培養(yǎng)24h、48h、72h后檢測相關(guān)指標(biāo)的變化。 (3)檢測指標(biāo)及其方法： ①采用反轉(zhuǎn)錄酶鏈聚合反應(yīng)(reverse transcription polymerase chain reaction, RT-PCR)實驗的方法檢測T24細(xì)胞中組織蛋白酶B的mRNA的表達(dá)。 ②采用組織蛋白酶B裂解底物的實驗方法來檢測T24細(xì)胞中的組織蛋白酶B的蛋白活性。 ③通過免疫細(xì)胞化學(xué)(immunocytochemical method)實驗的方法來檢測T24細(xì)胞中的組織蛋白酶B的蛋白表達(dá)。 ④應(yīng)用蛋白質(zhì)免疫印跡(Western blot)實驗的方法來檢測T24細(xì)胞中的組織蛋白酶B的蛋白的表達(dá)。 ⑤通過Transwell小室實驗的方法檢測T24細(xì)胞的遷移和侵襲。(4)統(tǒng)計學(xué)分析所有的實驗數(shù)據(jù)均通過SPSS17.0軟件處理,組間差異用單因素方差 分析的統(tǒng)計學(xué)方法進(jìn)行實驗數(shù)據(jù)處理。以α=0.05為檢驗標(biāo)準(zhǔn)。 結(jié)果 (1)T24細(xì)胞中組織蛋白酶B的mRNA的表達(dá)在各組間無明顯差異,不具有統(tǒng)計學(xué)意義(P0.05)。 (2)T24細(xì)胞中組織蛋白酶B的蛋白量的表達(dá)及活性的表達(dá)均隨CA-074濃度的增加及培養(yǎng)時間的延長而降低。與空白對照組對照組及實驗對照組比均有統(tǒng)計學(xué)意義(P0.05)。 (3)T24細(xì)胞隨著CA-074濃度的增加及培養(yǎng)時間的延長穿膜細(xì)胞數(shù)明顯減少具有統(tǒng)計學(xué)意義(P0.05)。 (4)各空白對照組和實驗對照組間各項指標(biāo)的檢測均不具有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 (1)CA-074對T24細(xì)胞的遷移和侵襲均具有明顯抑制作用。 (2)組織蛋白酶B可能參與了T24細(xì)胞的遷移和侵襲,并起到了一定的作用。
[Abstract]:Background bladder cancer is one of the most common malignant tumors in urology. Bladder urothelial carcinoma (bladder urothelial carcinoma, BUC) accounts for more than 90% of bladder cancer. At present, the treatment of bladder cancer is mainly surgery, but in recent years, the new molecular targeted therapy makes us see the hope of not treating tumor through surgery. Cathepsin B (cathepsin B, CB) belongs to papaya protein family and is an important sulfhydryl protease in lysosome. Recent studies have found that cathepsin B is associated with malignant phenotypes such as tumor invasion and metastasis. However, there is no in vitro study on the relationship between cathepsin B and T 24 cell migration and invasion. Objective to investigate the expression of cathepsin B in human bladder urothelial carcinoma T24 cells, and to reduce the expression and activity of cathepsin B protein by CA-074, a specific inhibitor of cathepsin B. The migration and invasiveness of T 24 cells were observed. Methods (1) Experimental preparation: purchase and subculture of T 24 cells. CA-074 was dissolved into required concentration by dimethyl sulfoxide (DMSO). (2) normal cultured T24 cells were divided into four experimental groups, and T24 cells cultured with different final concentrations of CA-074 were divided into 4 groups. The concentration of T24 cells was 0. 5 渭 mol/L, 1 渭 mol/L, 5 渭 mol/L and 10 渭 mol/L, respectively. T24 cells cultured in the medium containing solvent DMSO were used as the experimental control group. (3) Detection indexes and their methods: 1 Detection of cathepsin B in T24 cells by reverse transcriptase polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR) assay The protein activity of cathepsin B in T24 cells was detected by using cathepsin B substrates. 3 the activity of cathepsin B was detected by immunocytochemical (immunocytochemical method) assay. The protein expression of cathepsin B in T24 cells was measured. 4 the expression of cathepsin B protein in T24 cells was detected by Western blot (Western blot) assay. 5 the expression of cathepsin B protein was detected by Transwell chamber. The method was used to detect the migration and invasion of T24 cells. (4) all the experimental data were processed by SPSS17.0 software. The experimental data were processed by single factor ANOVA statistical method. 偽 = 0.05 was used as the test standard. Results (1) there was no significant difference in the expression of cathepsin B mRNA in T24 cells. The expression and activity of cathepsin B in T24 cells with no statistical significance (P0.05). (2) decreased with the increase of CA-074 concentration and the prolongation of culture time. Compared with the blank control group and the experimental control group, there was statistical significance (P0.05). (3) T24 cells decreased significantly with the increase of CA-074 concentration and the extension of culture time (P0.05). (4). There was no statistical significance between the blank control group and the experimental control group (P0.05). Conclusion (1) CA-074 can significantly inhibit the migration and invasion of T24 cells. (2) cathepsin B may play a role in the migration and invasion of T24 cells.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.14

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