Rce1在膀胱癌的表达及其对T24细胞周期和凋亡的影响
发布时间：2018-07-29 17:03
【摘要】：目的:检测Rce1(Ras and a-factor converting enzyme 1)在膀胱肿瘤组织及对应肿瘤旁正常移行上皮组织中的动态改变及其临床意义;以慢病毒为载体,构建shRNA干扰序列沉默Rce1基因,观察Rce1低表达对人膀胱癌T24细胞凋亡百分比的影响和细胞周期分布的改变。方法:采用实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)和蛋白印记杂交(western blot)技术,分别检测Rce1在12例膀胱肿瘤组织及对应肿瘤旁正常上皮组织中mRNA和蛋白表达差异;应用免疫组化技术,检测78例膀胱肿瘤组织,21例肿瘤旁正常上皮组织中Rce1的动态改变情况;结合病人的临床p TNM分期,细胞组织学分级等病理资料,分析Rce1的动态改变与病人临床特征,病理分型的关系。以慢病毒为载体,以Rce1为目的基因,设计shRNA-Rce1干扰序列,转染T24细胞,采用嘌呤霉素筛选,建立稳定低表达Rce1的T24细胞系;采用实时荧光定量PCR和western blot技术,分别检测各组T24细胞中Rce1在mRNA和蛋白的表达水平;应用流式细胞技术(flow cytometry,FCM),检测各组T24细胞在细胞周期的分布情况,细胞凋亡率所占百分比。结果:qRT-PCR和western blot显示,在12对膀胱肿瘤及肿瘤旁正常上皮组织中Rce1存在表达量的动态改变,差异有统计学意义(P=0.00);78例肿瘤组织中,Rce1阳性表达率为70.5%(55/78),肿瘤旁正常上皮组织中,检测到Rce1表达的比例为19.0%(4/21),Rce1阳性表达在肿瘤及肿瘤旁正常上皮组织的百分比存在差异,统计学分析差别有意义(P=0.00);统计学分析提示,Rce1阳性表达率在肿瘤的细胞组织学分级的各个分级百分比存在差异,在pTNM分期中各T分期中阳性表达率也有差异,Rce1在存在肿瘤远处转移的病人中阳性表达百分比跟高(P0.05);Kaplan-Meier分析不同病人的生存时间显示,与Rce1阴性病人相比,膀胱癌肿瘤中能够检测到Rce1表达的病人预后比较差(P=0.016)。与未感染病的空白对照组和感染病毒的阴性对照组相比,感染慢病毒干扰序列的T24细胞中Rce1在mRNA和蛋白表达水平明显下调(P=0.00),Rce1低表达T24细胞凋亡百分比增加,shRNA-Rce1组细胞在各周期的比例没有显著变化。结论:Rce 1在膀胱肿瘤组织中表达高于肿瘤旁正常上皮组织,Rce1可能参与了膀胱肿瘤的发生发展中;慢病毒干扰序列shRNA沉默Rce1基因能有效下调Rce1在T24细胞中表达,并诱导T24细胞凋亡;Rce1有望成为膀胱癌临床诊治和预后的一个新指标。
[Abstract]:Objective: to detect the dynamic changes and clinical significance of Rce1 (Ras and a-factor converting enzyme 1 in bladder tumor tissues and normal transitional epithelial tissues adjacent to the corresponding tumors, and construct shRNA interference sequence silencing Rce1 gene with lentivirus as vector. To observe the effect of low expression of Rce1 on the percentage of apoptosis and the change of cell cycle distribution in human bladder cancer cell line T24. Methods: the expression of mRNA and protein in 12 cases of bladder tumor and normal epithelium adjacent to bladder tumor were detected by real-time fluorescent quantitative PCR (Quantitative Real-time PCR and protein imprinting hybridization (western blot) technique, and the expression of mRNA and protein in 12 cases of bladder tumor tissues and normal epithelium adjacent to the corresponding tumor were detected by immunohistochemical technique. The dynamic changes of Rce1 in 21 cases of normal epithelial tissue adjacent to the tumor were detected, and the dynamic changes of Rce1 and the clinical features of the patients were analyzed in combination with the pathological data of clinical p TNM staging and histological grading. The relationship between pathological classification. Using lentivirus as vector and Rce1 as target gene, the interference sequence of shRNA-Rce1 was designed and transfected into T24 cells. A stable T24 cell line with low expression of Rce1 was established by purine mycin screening, and real-time fluorescence quantitative PCR and western blot techniques were used. The expression of Rce1 in mRNA and protein was detected in T24 cells of each group, and the distribution of T24 cells in cell cycle and the percentage of apoptosis were detected by flow cytometry (FCM). Results in 12 pairs of bladder tumors and adjacent normal epithelial tissues, the positive rate of Rce1 expression was 70.5% (55 / 78) and 70.5% (55 / 78), respectively. The percentage of positive expression of Rce1 was 19.0% (4 / 21). Statistical analysis showed that the positive expression rate of Rce1 was different in the percentage of tumor cell histological grade. The percentage of positive expression of Rce1 in patients with distant metastasis of tumor was higher than that in patients with Rce1 negative by Kaplan-Meier analysis. Patients with Rce1 expression in bladder cancer had poor prognosis (P0. 016). Compared with the uninfected blank control group and the viral negative control group, The percentage of apoptosis of T24 cells infected with lentivirus interference sequence decreased significantly in mRNA and protein expression (P0. 00). The percentage of T24 cells with low expression of Rce1 increased, and the percentage of T24 cells in each cell cycle did not change significantly. Conclusion the expression of RCE 1 in bladder tumor tissue is higher than that in normal epithelial tissue adjacent to the tumor, and the lentivirus interference sequence shRNA silencing Rce1 gene can effectively down-regulate the expression of Rce1 in T24 cells. The induction of T24 cell apoptosis and Rce1 may be a new marker for the diagnosis, treatment and prognosis of bladder cancer.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14
[Abstract]:Objective: to detect the dynamic changes and clinical significance of Rce1 (Ras and a-factor converting enzyme 1 in bladder tumor tissues and normal transitional epithelial tissues adjacent to the corresponding tumors, and construct shRNA interference sequence silencing Rce1 gene with lentivirus as vector. To observe the effect of low expression of Rce1 on the percentage of apoptosis and the change of cell cycle distribution in human bladder cancer cell line T24. Methods: the expression of mRNA and protein in 12 cases of bladder tumor and normal epithelium adjacent to bladder tumor were detected by real-time fluorescent quantitative PCR (Quantitative Real-time PCR and protein imprinting hybridization (western blot) technique, and the expression of mRNA and protein in 12 cases of bladder tumor tissues and normal epithelium adjacent to the corresponding tumor were detected by immunohistochemical technique. The dynamic changes of Rce1 in 21 cases of normal epithelial tissue adjacent to the tumor were detected, and the dynamic changes of Rce1 and the clinical features of the patients were analyzed in combination with the pathological data of clinical p TNM staging and histological grading. The relationship between pathological classification. Using lentivirus as vector and Rce1 as target gene, the interference sequence of shRNA-Rce1 was designed and transfected into T24 cells. A stable T24 cell line with low expression of Rce1 was established by purine mycin screening, and real-time fluorescence quantitative PCR and western blot techniques were used. The expression of Rce1 in mRNA and protein was detected in T24 cells of each group, and the distribution of T24 cells in cell cycle and the percentage of apoptosis were detected by flow cytometry (FCM). Results in 12 pairs of bladder tumors and adjacent normal epithelial tissues, the positive rate of Rce1 expression was 70.5% (55 / 78) and 70.5% (55 / 78), respectively. The percentage of positive expression of Rce1 was 19.0% (4 / 21). Statistical analysis showed that the positive expression rate of Rce1 was different in the percentage of tumor cell histological grade. The percentage of positive expression of Rce1 in patients with distant metastasis of tumor was higher than that in patients with Rce1 negative by Kaplan-Meier analysis. Patients with Rce1 expression in bladder cancer had poor prognosis (P0. 016). Compared with the uninfected blank control group and the viral negative control group, The percentage of apoptosis of T24 cells infected with lentivirus interference sequence decreased significantly in mRNA and protein expression (P0. 00). The percentage of T24 cells with low expression of Rce1 increased, and the percentage of T24 cells in each cell cycle did not change significantly. Conclusion the expression of RCE 1 in bladder tumor tissue is higher than that in normal epithelial tissue adjacent to the tumor, and the lentivirus interference sequence shRNA silencing Rce1 gene can effectively down-regulate the expression of Rce1 in T24 cells. The induction of T24 cell apoptosis and Rce1 may be a new marker for the diagnosis, treatment and prognosis of bladder cancer.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.14
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