组织蛋白酶B对人膀胱尿路上皮癌T24细胞迁移和侵袭的影响
发布时间：2018-10-21 10:47
【摘要】：背景 膀胱癌是我国泌尿外科临床工作中最常见的恶性肿瘤中的一种。膀胱尿路上皮癌(bladder urothelial carcinoma, BUC)在膀胱癌中占的比例大约90%以上。目前膀胱癌的治疗以手术为主,但近几年新兴的分子靶向治疗,使我们看到了不通过手术治疗肿瘤的希望。组织蛋白酶B (cathepsin B, CB)属于木瓜蛋白家族,是溶酶体内重要的一种巯基蛋白酶。近年来的研究发现组织蛋白酶B与肿瘤的侵袭和转移等恶性表型相关。但尚无组织蛋白酶B与T24细胞迁移和侵袭相关性的体外研究。 目的 探讨组织蛋白酶B在人膀胱尿路上皮癌T24细胞中的表达情况；通过组织蛋白酶B的特异性抑制剂CA-074降低组织蛋白酶B蛋白的表达及活性,观察T24细胞的迁移和侵袭力的变化。 方法 (1)实验准备：购买并传代培养实验用T24细胞。将CA-074用二甲基亚砜(DMSO)溶解成需要浓度。 (2)实验分组：正常培养的T24细胞为空白对照组；以不同终浓度的CA-074培养液培养的T24细胞分为4个实验组,分别为0.5μ mol/L浓度组、1μ mol/L浓度组、5μ mol/L浓度组、10μ mol/L浓度组；以含有溶剂DMSO的培养基培养的T24细胞作为实验对照组。分别培养24h、48h、72h后检测相关指标的变化。 (3)检测指标及其方法： ①采用反转录酶链聚合反应(reverse transcription polymerase chain reaction, RT-PCR)实验的方法检测T24细胞中组织蛋白酶B的mRNA的表达。 ②采用组织蛋白酶B裂解底物的实验方法来检测T24细胞中的组织蛋白酶B的蛋白活性。 ③通过免疫细胞化学(immunocytochemical method)实验的方法来检测T24细胞中的组织蛋白酶B的蛋白表达。 ④应用蛋白质免疫印迹(Western blot)实验的方法来检测T24细胞中的组织蛋白酶B的蛋白的表达。 ⑤通过Transwell小室实验的方法检测T24细胞的迁移和侵袭。(4)统计学分析所有的实验数据均通过SPSS17.0软件处理,组间差异用单因素方差 分析的统计学方法进行实验数据处理。以α=0.05为检验标准。 结果 (1)T24细胞中组织蛋白酶B的mRNA的表达在各组间无明显差异,不具有统计学意义(P0.05)。 (2)T24细胞中组织蛋白酶B的蛋白量的表达及活性的表达均随CA-074浓度的增加及培养时间的延长而降低。与空白对照组对照组及实验对照组比均有统计学意义(P0.05)。 (3)T24细胞随着CA-074浓度的增加及培养时间的延长穿膜细胞数明显减少具有统计学意义(P0.05)。 (4)各空白对照组和实验对照组间各项指标的检测均不具有统计学意义(P0.05)。 结论 (1)CA-074对T24细胞的迁移和侵袭均具有明显抑制作用。 (2)组织蛋白酶B可能参与了T24细胞的迁移和侵袭,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. 伪 = 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.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14
本文编号：2284865
[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. 伪 = 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.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14
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