【摘要】：目的:檢測Rce1(Ras and a-factor converting enzyme 1)在膀胱腫瘤組織及對應腫瘤旁正常移行上皮組織中的動態(tài)改變及其臨床意義;以慢病毒為載體,構(gòu)建shRNA干擾序列沉默Rce1基因,觀察Rce1低表達對人膀胱癌T24細胞凋亡百分比的影響和細胞周期分布的改變。方法:采用實時熒光定量PCR(Quantitative Real-time PCR,qRT-PCR)和蛋白印記雜交(western blot)技術(shù),分別檢測Rce1在12例膀胱腫瘤組織及對應腫瘤旁正常上皮組織中mRNA和蛋白表達差異;應用免疫組化技術(shù),檢測78例膀胱腫瘤組織,21例腫瘤旁正常上皮組織中Rce1的動態(tài)改變情況;結(jié)合病人的臨床p TNM分期,細胞組織學分級等病理資料,分析Rce1的動態(tài)改變與病人臨床特征,病理分型的關(guān)系。以慢病毒為載體,以Rce1為目的基因,設計shRNA-Rce1干擾序列,轉(zhuǎn)染T24細胞,采用嘌呤霉素篩選,建立穩(wěn)定低表達Rce1的T24細胞系;采用實時熒光定量PCR和western blot技術(shù),分別檢測各組T24細胞中Rce1在mRNA和蛋白的表達水平;應用流式細胞技術(shù)(flow cytometry,FCM),檢測各組T24細胞在細胞周期的分布情況,細胞凋亡率所占百分比。結(jié)果:qRT-PCR和western blot顯示,在12對膀胱腫瘤及腫瘤旁正常上皮組織中Rce1存在表達量的動態(tài)改變,差異有統(tǒng)計學意義(P=0.00);78例腫瘤組織中,Rce1陽性表達率為70.5%(55/78),腫瘤旁正常上皮組織中,檢測到Rce1表達的比例為19.0%(4/21),Rce1陽性表達在腫瘤及腫瘤旁正常上皮組織的百分比存在差異,統(tǒng)計學分析差別有意義(P=0.00);統(tǒng)計學分析提示,Rce1陽性表達率在腫瘤的細胞組織學分級的各個分級百分比存在差異,在pTNM分期中各T分期中陽性表達率也有差異,Rce1在存在腫瘤遠處轉(zhuǎn)移的病人中陽性表達百分比跟高(P0.05);Kaplan-Meier分析不同病人的生存時間顯示,與Rce1陰性病人相比,膀胱癌腫瘤中能夠檢測到Rce1表達的病人預后比較差(P=0.016)。與未感染病的空白對照組和感染病毒的陰性對照組相比,感染慢病毒干擾序列的T24細胞中Rce1在mRNA和蛋白表達水平明顯下調(diào)(P=0.00),Rce1低表達T24細胞凋亡百分比增加,shRNA-Rce1組細胞在各周期的比例沒有顯著變化。結(jié)論:Rce 1在膀胱腫瘤組織中表達高于腫瘤旁正常上皮組織,Rce1可能參與了膀胱腫瘤的發(fā)生發(fā)展中;慢病毒干擾序列shRNA沉默Rce1基因能有效下調(diào)Rce1在T24細胞中表達,并誘導T24細胞凋亡;Rce1有望成為膀胱癌臨床診治和預后的一個新指標。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R737.14

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