【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(shí)驗(yàn)準(zhǔn)備：購買并傳代培養(yǎng)實(shí)驗(yàn)用T24細(xì)胞。將CA-074用二甲基亞砜(DMSO)溶解成需要濃度。 (2)實(shí)驗(yàn)分組：正常培養(yǎng)的T24細(xì)胞為空白對(duì)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(gè)實(shí)驗(yàn)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(shí)驗(yàn)對(duì)照組。分別培養(yǎng)24h、48h、72h后檢測(cè)相關(guān)指標(biāo)的變化。 (3)檢測(cè)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(shí)驗(yàn)的方法檢測(cè)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(shí)驗(yàn)方法來檢測(cè)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(shí)驗(yàn)的方法來檢測(cè)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(shí)驗(yàn)的方法來檢測(cè)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(shí)驗(yàn)的方法檢測(cè)T24細(xì)胞的遷移和侵襲。(4)統(tǒng)計(jì)學(xué)分析所有的實(shí)驗(yàn)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(jì)學(xué)方法進(jìn)行實(shí)驗(yàn)數(shù)據(jù)處理。以α=0.05為檢驗(yàn)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(jì)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(shí)間的延長而降低。與空白對(duì)照組對(duì)照組及實(shí)驗(yàn)對(duì)照組比均有統(tǒng)計(jì)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(shí)間的延長穿膜細(xì)胞數(shù)明顯減少具有統(tǒng)計(jì)學(xué)意義(P0.05)。 (4)各空白對(duì)照組和實(shí)驗(yàn)對(duì)照組間各項(xiàng)指標(biāo)的檢測(cè)均不具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論 (1)CA-074對(duì)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(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 段建敏,左陵君,范文高,秦大山,段國蘭;組織蛋白酶B與膀胱癌惡性度的相關(guān)性[J];臨床泌尿外科雜志;2003年11期

2 段建敏,高棟,張建平,秦大山;腎癌患者組織蛋白酶B檢測(cè)的臨床意義[J];蘭州醫(yī)學(xué)院學(xué)報(bào);2003年01期

3 周國宏;于文貞;黎曉新;何培英;董建強(qiáng);;CA-074Me對(duì)體外類毛細(xì)血管形成的影響[J];眼科研究;2007年06期

4 張薇,項(xiàng)永兵,劉振偉,方茹蓉,阮志賢,孫璐,高立峰,金凡,高玉堂;1973-1999年上海市區(qū)老年人惡性腫瘤發(fā)病趨勢(shì)分析[J];中華老年醫(yī)學(xué)雜志;2005年09期

5 魏礦榮,陳振雄,梁智恒,何方方,林茂合,歐小文;中山市1970～1999年膀胱癌發(fā)病趨勢(shì)分析[J];中國腫瘤;2005年04期

6 陳必成,吳秀玲,張方毅,李澄棣;Survivin在膀胱移行細(xì)胞癌中的表達(dá)及意義[J];浙江醫(yī)學(xué);2002年06期

相關(guān)博士學(xué)位論文 前2條

1 肖虹;ALA光動(dòng)力診斷與光動(dòng)力治療惡性腦腫瘤的療效及機(jī)制研究[D];第三軍醫(yī)大學(xué);2009年

2 朱海斌;siRNA干擾突變型p53對(duì)泌尿系統(tǒng)腫瘤的治療作用及其機(jī)制研究[D];浙江大學(xué);2011年

，

本文編號(hào)：2284865