谷氨酰半胱氨酸连接酶在膀胱癌中表达的研究

发布时间：2018-05-05 00:43

本文选题：膀胱尿路上皮癌 + 谷胱甘肽　；参考：《山东大学》2014年博士论文

【摘要】：背景膀胱癌属于泌尿系统中最常见的一种的恶性肿瘤,近年来,膀胱尿路上皮癌的发病率和死亡率均有逐年增高的趋势,膀胱癌(bladder cancer)已成为危胁人类健康的世界性问题,在全球恶性肿瘤的发病率中位列第九位[1]。2006年美国癌症协会统计膀胱癌在男性中排名第四位,排在前列腺癌、肺癌和直肠癌之后。它是我国泌尿外科临床上最常见的恶性疾病之一,是一种对患者生命安全造成直接威胁的疾病。国内外学者对膀胱癌的危险因素做了大量的研究,其具体的发生病因目前还不清楚,有机化学致癌物和人体色氨酸,代谢异常是目前公认的常见原因。有研究发现膀胱长期残余尿、慢性膀胱炎、以及长期的膀胱结石、导管刺激与膀胱癌的发生有着密切关系[2]。膀胱癌的病理类型包括尿路移行上皮细胞癌、鳞状上皮细胞癌、腺细胞癌以及转移癌、癌肉瘤等,而移性上皮细胞癌为膀胱癌中最常见的病理类型。非上皮性膀胱肿瘤来自于膀胱间叶组织,临床上更为少见。膀胱癌的恶性程度以目前世界卫生组织(World Health Organization,WHO)分级法表示,分级与肿瘤的恶性程度成正比,肿瘤分期分级愈高,数目越多,体积愈大,术后肿瘤浸润和复发的可能性愈大。膀胱癌的临床分期是评判肿瘤预后最重要的参数,它与肿瘤浸润深度和有无转移密切相关。膀胱癌患者临床上多表现为无痛性肉眼血尿,可间歇性出现,如果伴有血块可造成急性尿潴留。晚期膀胱癌患者由于肿瘤浸润、转移可发生贫血、下腹部肿物、腰骶部疼痛、患肢水肿等临床症状。非肌层浸润性膀胱癌可行经尿道膀胱肿瘤切除术,术后辅以局部膀胱灌洗化疗。其发病机理还不完全明了。近年来,已经有大量的数据研究发现,在机体细胞恶性增殖导致肿瘤产生的过程中,体内的氧化还原系统较正常状态下发生不同的改变,氧化还原状态的失衡可能在肿瘤的病理进展中非常重要,这与氧化应激导致细胞中的大分子脱氧核糖核酸功能受损有一定的关系。各种氧化产物与恶性疾病的病情发展密切相关,甚至,在癌症发生以前就可能出现自由基和活性氧产物增多的现象。在各种不同的恶性组织细胞中,抗氧化能力的下降导致的后果是机体内产生各种不同的大量的活性氧产物。在膀胱尿路上皮癌中,机体内的的氧化应激状态发生了不同的改变[5]。在膀胱癌中,部分与氧化系统密切有关的物质例如脂质、蛋白及DNA等均产生不同程度的变化[6]。有学者研究认为活性氧成分在黑色素瘤的疾病进展过程中发挥十分显著的作用,针对活性氧成分的治疗措施能够显著的抑制癌细胞和抑制疾病的进展。谷胱甘肽作为机体清除自由基的最重要的抗氧化成分之一在机体的各种防御系统中扮演着重要的角色,谷胱甘肽作为体内相当显著的抗氧化成分,对保护机体各种组织细胞中的蛋白质和酶类分子中的巯基发挥相当重要的作用。谷氨酰半胱氨酸连接酶(GCL)是谷胱甘肽GSH在代谢中发挥重要作用的关键酶,其亚基在基因水平和蛋白水平及活性水平的改变能够使恶性增殖细胞中的活性氧产物含量改变同时影响肿瘤细胞增殖的进程。目的目前,谷氨酰半胱氨酸连接酶作为谷胱甘肽代谢过程中的关键酶在膀胱尿路上皮癌中尚无系统性的研究,我们希望通过对谷氨酰半胱氨酸连接酶在基因水平、蛋白水平和活性水平中较为系统的研究,探讨其在膀胱尿路上皮癌中变化,为膀胱尿路上皮癌的诊断、治疗和预后分析提供有效的数据基础。方法对膀胱尿路上皮癌和正常对照人群的一般血液学指标、临床生化指标和出凝血数据等实验室检测项目进行了系统的统计学处理；方法对46例膀胱尿路上皮癌组织和39例对照癌旁组织中的谷氨酰半胱氨酸连接酶的催化亚基(GCLC)和调节亚基(GCLM)信使核糖核酸(mRNA)的表达情况运用实时荧光定量PCR(quantitative realtime-PCR)进行实验分析；方法对膀胱尿路上皮癌组织和对照癌旁组织中的谷氨酰半胱氨酸连接催化亚基和调节亚基蛋白表达含量使用蛋白印迹(Western blotting)进行实验分析；运用酶的活性实验方法分析膀胱尿路上皮癌组织和对照癌旁组织中的谷氨酰半胱氨酸连接活性的改变。在实验过程中所收集的数据通过SPSS13.0统计学软件进行分析处理,采用Student's two-tailed t-tests方法进行差异显著性分析,p0.05为差异具有显著性,统计数据均采用均数±标准误(SEM)方式来表示。结果荧光定量PCR的实验结果分析显示,与癌旁组织的对照组相比较,膀胱尿路上皮癌组织中谷氨酰半胱氨酸连接酶的催化亚基GCLc和调节亚基GCLm基因表达出现显著降低的变化,两者之间具有显著的统计学差异(GCLc基因：BUC组1±0.028,对照组3.74±0.545;p0.01)(GCLm基因：BUC组1±0.05,对照组3.07±0.571；p0.01)。根据蛋白印迹的的实验结果分析显示,与癌旁组织的对照组相比较,在膀胱尿路上皮癌组织中谷氨酰半胱氨酸连接酶的催化亚基和调节亚基的蛋白表达显示显著下降的变化,两者之间具有显著的统计学差异(GCLc基因：BUC组0.983±0.022,对照组1.248±0.076;p0.05)(GCLm基因：BUC组1.103±0.014,对照组1.306±0.033；p0.05)。根据酶活性的实验结果分析显示,与癌旁组织的对照组相比较,谷氨酰半胱氨酸连接酶的活性在膀胱尿路上皮癌组织中的活性亦出现显著下降的趋势(p0.01)。膀胱尿路上皮癌中GCL的平均酶活为98+21mmol/分钟/mg蛋白而对照组织中的GCL平均酶活为。342±85mmol/分钟/mg蛋白。结论在膀胱尿路上皮癌中,谷氨酰半胱氨酸连接酶催化亚基及调节亚基无论在基因表达还是蛋白含量均明显降低,提示在膀胱尿路上皮癌中可能通过谷氨酰半胱氨酸连接酶基因表达和蛋白含量的变化进而调节谷胱甘肽在肿瘤细胞中的表达,从而对肿瘤细胞的氧化状态造成影响,进而影响肿瘤细胞的增殖和分化；膀胱尿路上皮癌中GCL酶活性显著升高,提示在肿瘤细胞中,可能因为酶含量的变化需要更高的酶活性来调节细胞的氧化平衡状态。本研究结果显示谷胱甘肽代谢过程中的限速酶谷氨酰半胱氨酸连接酶可能作为治疗膀胱尿路上皮癌及判断膀胱尿路上皮癌患者预后的一个有效的靶点。
[Abstract]:background
Bladder cancer is one of the most common malignant tumors in the urinary system. In recent years, the incidence and mortality of bladder urothelial carcinoma have been increasing year by year. Bladder cancer (bladder cancer) has become a worldwide problem threatening human health. It is the ninth American Cancer Association in [1].2006 years in the incidence of global malignant tumor. Statistical bladder cancer ranked fourth in the male, after prostate cancer, lung cancer and rectal cancer. It is one of the most common malignant diseases in the Department of Urology in our country. It is a direct threat to the life safety of the patients. The domestic and foreign scholars have done a lot of research on the risk factors of bladder cancer, and the specific cause of the disease. It is not clear that organic chemical carcinogens and human tryptophan and metabolic abnormalities are commonly recognized causes. Studies have found long-term residual urinary bladder, chronic cystitis, and long-term bladder stones. Catheter stimulation is closely related to the occurrence of bladder cancer, which is closely related to the pathological types of [2]. bladder cancer, including urinary transitional epithelial cell carcinoma, scales. Epithelial cell carcinoma, adenocarcinoma, metastatic carcinoma and carcinosarcoma are the most common pathological types of bladder cancer. Non epithelial bladder tumor is from the intravesical leaf tissue and is more rare in clinic. The malignancy of bladder cancer is expressed by the current WHO (World Health Organization, WHO) classification method. The higher the malignancy of the tumor, the higher the stage of the tumor, the more the number, the larger the volume, the greater the possibility of the invasion and recurrence of the tumor. The clinical stage of bladder cancer is the most important parameter to judge the prognosis of the tumor. It is closely related to the depth of invasion and the metastasis of the tumor. Ocular hematuria, which may occur intermittently, can cause acute urinary retention if accompanied by blood clot. Patients with advanced bladder cancer may have clinical symptoms such as anemia, abdominal mass, lumbosacral pain, and limb edema due to tumor infiltration. Non muscular invasive bladder cancer can be excised by urethral bladder tumor and adjuvant chemotherapy after local bladder irrigation. The pathogenesis is not completely clear. In recent years, a large number of data have been found that the redox system in the body is different from the normal state in the process of the malignant proliferation of the body, and the imbalance of redox state may be very important in the pathological progress of the tumor, which is related to the oxidative stress. There is a certain relationship between the damage to the function of the large molecular deoxyribonucleic acid in the cells. Various oxidation products are closely related to the development of the disease. Even the phenomenon of the increase of free radicals and reactive oxygen species may occur before the occurrence of cancer. The consequences of the decline in antioxidant capacity in various malignant tissue cells A variety of active oxygen products are produced in the body. In bladder urothelial carcinoma, the oxidative stress in the body has different changes in the state of [5]. in bladder cancer. Some substances such as lipid, protein, and DNA, which are closely related to the oxidation system, have varying degrees of change. [6]. has been studied for reactive oxygen species. Ingredients play a significant role in the progression of melanoma, and the treatment of reactive oxygen species can significantly inhibit the progression of cancer cells and inhibition of disease. Glutathione, as one of the most important antioxidant components in the body, plays an important role in the various defense systems of the body, Glutathione, a very significant antioxidant in the body, plays an important role in protecting the sulfhydryl groups of proteins and enzymes in various tissues and cells. GCL is a key enzyme that plays an important role in the metabolism of glutathione GSH. Its subunits are at gene level, protein level and activity. The change of sex level can change the content of reactive oxygen species in malignant proliferative cells and influence the process of tumor cell proliferation.
objective
At present, there is no systematic study of the glutamine cysteine ligase as a key enzyme in the metabolic process of glutathione. We hope to explore the changes in the bladder urothelial carcinoma by systematic study of the glutamine cysteine ligase at the level of gene, protein and activity. The diagnosis, treatment and prognosis analysis of bladder urothelial carcinoma provide an effective data base.
Method
The general hematological indexes, clinical biochemical indexes and blood coagulation data in normal controls were systematically treated. The catalytic subunit (GCLC) and regulation of glutamyl cysteinic ligase (GCLC) in 39 cases of bladder urothelial carcinoma and 39 control para cancerous tissues were treated. The expression of GCLM messenger RNA (mRNA) was analyzed by real time fluorescence quantitative PCR (quantitative realtime-PCR). The expression of glutamyl cysteine linked catalytic subunit and regulatory subunit protein in the bladder urothelial carcinoma tissue and the control para cancerous tissue was used to use Western blot (Western blotting). The changes in the activity of glutamyl cysteine connection in the bladder urothelial carcinoma and the control of the para cancerous tissue were analyzed by the enzyme activity test. The data collected during the experiment were analyzed by the SPSS13.0 statistical software, and the difference was statistically significant by the Student's two-tailed t-tests method. Analysis shows that P0.05 is significant for difference, and statistical data are expressed by means of mean + standard error (SEM).
Result
The results of the fluorescence quantitative PCR analysis showed that the catalytic subunit GCLc and the regulatory subunit GCLm gene expression in the bladder urothelial carcinoma were significantly lower than those in the control group of the para cancerous tissue, and there were significant differences between the two groups (GCLc gene: the BUC group was 1 + 0.028, and the control group was 3. .74 + 0.545; P0.01) (GCLm gene: BUC group 1 + 0.05, control group 3.07 + 0.571; P0.01). According to the experimental results of Western blot analysis, the expression of the protein expression of the catalytic subunit and the regulatory subunit of the glutamyl cysteine ligase in the bladder urothelial carcinoma tissue was significantly decreased. There were significant statistical differences between the two (GCLc gene: group BUC 0.983 + 0.022, control group 1.248 + 0.076; P0.05) (GCLm gene: BUC group 1.103 + 0.014, control group 1.306 + 0.033; P0.05). According to experimental results of enzyme activity, the activity of glutamyl cysteine ligase in bladder urine was compared with the control group of paracancerous tissue. The activity in the skin carcinoma of the road was also significantly decreased (P0.01). The average enzyme activity of GCL in bladder urothelial carcinoma was 98+21mmol/ min /mg protein, while the average GCL enzyme activity in the control tissue was.342 + 85mmol/ minutes /mg protein.
conclusion
In bladder urothelial carcinoma, the glutamyl cysteine ligase catalyzed subunit and the regulatory subunit decreased significantly in gene expression and protein content, suggesting that the expression of glutamyl cysteine ligase gene and protein content may be regulated in bladder urothelial carcinoma to regulate the table of glutathione in tumor cells. It has an effect on the oxidation state of tumor cells, and then affects the proliferation and differentiation of tumor cells. The activity of GCL enzyme in bladder urothelial carcinoma increases significantly. It suggests that in the tumor cells, higher enzyme activity may be needed to regulate the state of oxygen balance in the tumor cells. The results of this study show that the glutathione generation is glutathione. The speed limiting enzyme glutamyl cysteine ligase in Xie Guocheng may be an effective target for the treatment of bladder urothelial carcinoma and the prognosis of bladder urothelial carcinoma.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.14

【共引文献】