【作者】 杨小清；

【导师】 韩瑞发；

【作者基本信息】 天津医科大学 ， 外科学， 2014， 博士

【摘要】 第一部分膀胱癌合并糖尿病患者的临床、病理特点目的：分析总结中国人群中膀胱癌合并糖尿病患者的临床特点以及肿瘤的组织学特点。方法：收集并统计天津医科大学第二医院泌尿外科2008年1月-2011年12月期间所有新发的膀胱癌患者的诊治病例资料,共计992份,将这些膀胱癌患者按照是否合并糖尿病分为单纯膀胱癌组和膀胱癌合并糖尿病组,对两组患者的临床病理特点进行比较分析。结果：两组比较,膀胱癌合并糖尿病组患者体重、BMI明显高于单纯膀胱癌组(P=0.000、P=0.000)。膀胱癌合并糖尿病组患者FPG水平明显高于单纯膀胱癌组(P=0.000),且膀胱癌合并糖尿病组患者合并高血压病的比例更高(P=0.007)。与单纯膀胱组患者相比较,膀胱癌合并糖尿病组患者肿瘤更多发生于膀胱颈部位(P=0.002),肿瘤病理分级的级别更高(P=0.042),初诊时即发生远处转移的比例更高(P=0.000)。两组间患者性别、是否吸烟、肿瘤分期、肿瘤大小、肿瘤数目、初诊时淋巴结转移等情况差异无统计学意义。结论：糖尿病增加膀胱癌患者的体重、BMI；与单纯膀胱癌组患者相比较,膀胱癌合并糖尿病组患者血糖水平更高,且糖尿病增加膀胱癌患者的高血压合并率；与单纯膀胱癌组患者相比较,膀胱癌合并糖尿病组患者肿瘤更多见于膀胱颈部位,且膀胱癌合并糖尿病组患者的肿瘤病理分级别更高、初诊时即有较多患者伴有远处转移,表明糖尿病可能增加膀胱癌的恶性度。第二部分GLUT1在人膀胱尿路上皮癌组织中的表达及其与HIF1a、肿瘤血管生成的相关性研究目的：通过研究GLUT1在膀胱尿路上皮癌组织中的表达,探索糖尿病合并膀胱癌患者组织中的GLUT1的异常表达与肿瘤血管生成的相关性及调控机制。方法：应用免疫组化技术检测膀胱尿路上皮癌组织中GLUT1、HF1a、MVD、 VEGF的表达,比较单纯膀胱癌和合并糖尿病的膀胱癌组织中各指标的差异,分析GLUT1与HIF1a、MVD、VEGF的相关性。结果：GLUT1在癌旁正常膀胱组织中不表达,在UCC组织中过表达,且GLUT1的表达与肿瘤的分期、分级有关(P=0.000、P=0.000)。膀胱癌合并糖尿病组患者组织中GLUT1的表达水平高于单纯膀胱癌组(P=0.015)；HIFla在UCC组织中的表达与肿瘤的分期、分级有关(P=0.000、P=0.000),且与是否合并糖尿病有关(P=0.015)；MVD、VEGF在UCC组织中的表达与肿瘤的分期、分级有关(P=0.000、P=0.001; P=0.000、P=0.039),与是否合并糖尿病无关(P>0.05；P>0.05)；在UCC组织中,GLUT1的表达与HIF1a、MVD、VEGF呈正相关关系(r=0.794,P=0.000; r=0.549, P=0.006; r=0.381, P=0.006)。结论：GLUT1在UCC组织中的表达与肿瘤的分期、分级、肿瘤的缺氧微环境及肿瘤的血管形成有关,且糖尿病会增加UCC患者肿瘤组织中GLUT1的表达水平。第三部分GLUT1shRNA干扰对膀胱癌T24细胞株的效应研究目的：通过研究GLUT1对膀胱癌细胞的效应,探索GLUT1在膀胱癌进展中的作用。方法：通过体外细胞学实验研究利用重组质粒载体转染技术干扰膀胱癌细胞GLUT1水平,Q-PCR、western blotting检测RNA干扰效果,MTT、流式细胞术、Transwell等方法验证GLUT1对臃胱肿瘤细胞增殖、凋亡、周期以及侵袭的效应作用,并应用2-脱氧-D-[3H]-葡萄糖法检测细胞内葡萄糖摄取率的变化。结果：应用肿瘤细胞A549筛选出对GLUT1干扰效果达到82%的GLUT1-shRNA;MTT、流式细胞术、Transwell等结果显示,GLUT1-shRNA可抑制T24细胞的增殖达58.69%(48h)；促进T24细胞坏死凋亡,48h坏死和凋亡率为47.41%和22.03%；抑制T24细胞的侵袭能力。同时GLUT1-shRNA可减少T24细胞内葡萄糖摄取率达62.39%。结论：GLUT1在人肿瘤细胞包括人膀胱癌细胞中表达,并成功构建、筛选出对GLUT1干扰效果达到82%的pYr-1.1-GLUT1-shRNA质粒；体外实验证实GLUT1-shRNA可减少膀胱癌T24的细胞内葡萄糖摄取、抑制增殖、促进凋亡、抑制侵袭的作用。提示GLUT1可能在膀胱癌进展中发挥重要作用。第四部分GLUT1基因多态性与膀胱尿路上皮癌发病风险以及P53、Ki67的关系目的：GLUT1是肿瘤葡萄糖代谢的关键因素,探索GLUT1基因的单核苷酸多态性在UCC肿瘤形成和侵袭中作用。方法：选取UCC患者314例和健康对照者204例,提取血样DNA,应用PCR-RFLP方法进行GLUT1基因多态性分析,同时选取51例UCC患者的组织标本进行GLUT1、P53、Ki67免疫组化染色,分析GLUT1基因多态性与UCC发病风险以及GLUT1、P53、Ki67免疫组化结果的相关性。结果：与对照组相比,实验组中GLUT1XbaI G>T的TT基因型频率更低(38%vs.53％,P=0.033)；GT基因型频率更高(58%vs.43%,P=0.034)),T等位基因的分布频率更低(64%vs.74%,P=0.022,两组间GG基因型的频率差别没有统计学意义；实验组中GLUT1HaeⅢT>C的CC基因型频率更低(38%vs.53%,P=0.033),TC基因型频率更高(83%vs.73%,P=0.006),C等位基因的分布频率更低(45%vs.56%,P=0.001);两组间TT基因型的频率差别没有统计学意义。与低分期肿瘤患者相比较,高肿瘤分期患者中GLUT1XbaⅠ的GG基因型的频率更高(P=0.014)。与XbaⅠ G>T中CT/TT基因型相比,GG基因型患者组织中ki67(P=0.031)、GLUT1(P=0.009)的表达水平更高；XbaⅠ G>T各基因型中P53的表达差异没有统计学意义P=0.935)。HaeⅢ T>C各基因型中P53(P=0.964)、ki67(P=0.145),、GLUT1(P=0.167)的表达没有明显差异。结论：GLUT1XbaⅠ G>T中TT基因型和HaeⅢ T>C中CC基因型可能在UCC患者肿瘤发生中发挥保护作用；同时XbaⅠ G>T中GG基因型可能与肿瘤的增殖、肿瘤葡萄糖的摄取、肿瘤的恶性程度正相关。这些结果提示GLUT1可能会成为膀胱尿路上皮癌患者一个更高恶性度和更高增殖活性预测因素。更多还原

【Abstract】 Part I The clinical and pathological features of patients with bladder cancer associated with diabetes mellitusObjectives:Analyze and summarize the clinical features and histological features of the tumor of Chinese population of patients with bladder cancer associated with diabetes mellitus (DM).Methods:The clinical information of the patients with bladder cancer which was treated in the Department of Urology, Second Hospital of Tianjin Medical University in January2008-December2011period were collected, a total of992. The patients with bladder cancer were divided into bladder cancer with DM group and bladder cancer without DM group. The clinical and pathological characteristics of the two groups of patients were analyzed.Results:Comparing the two groups, the body weight and BMI was significantly higher in bladder cancer with DM group than bladder cancer without DM group (P=0.000、P=0.000). The level of FPG was significantly higher in bladder cancer with DM group than bladder cancer without DM group (P=0.000). The proportion of patients with hypertension was significantly higher in bladder cancer with DM group than bladder cancer without DM group (P=0.001). Compared with bladder cancer without DM group, the tumors were more occurred in bladder neck (P=0.002), the tumor grade was higher (P=0.042), the proportion of distant metastases was higher (P=0.000) in bladder cancer with DM group. No significant difference was seen between two groups when the gender, smoking, tumor stage, tumor size, tumor numbers and lymph node metastasis were assessed.Conclusion:DM increases the body weight and BMI of patients with bladder cancer. Compared with bladder cancer without DM group, the level of FPG and the proportion of patients with hypertension were significantly higher in bladder cancer with DM group. The tumors were more occurred in bladder neck and the tumor grade was higher in bladder cancer with DM group. The higher proportion of distant metastases in bladder cancer with DM means that DM may increase the degree of malignancy of bladder cancer. Part Ⅱ The expression of GLUT1in tumor tissue in UCC and it correlated research with HIFla and angiogenesisObjectives:By studying the expression of GLUT1in urothelial cell carcinomas of the bladder (UCC), to explore the abnormal expression of GLUT1in UCC with DM, and it correlation with tumor angiogenesis and regulatory mechanisms.Methods:The expression of GLUT1、HIF1a、MVD and VEGF was detected in UCC by immunohistochemistry. Differences in each index were compared between bladder cancer with DM group and bladder cancer without DM group. The correlation between GLUT1and HIF1a、MVD. VEGF was analyzed.Results:GLUT1was not expressed in adjacent normal bladder tissues, but was overexpressed in UCC tissues. And the expression of GLUT1was correlated with tumor staging (P=0.000) and grading (P=0.000). The expression of GLUT1was significantly higher in UCC with DM than UCC without DM (P=0.015). The expression of HIF1a was correlated with tumor staging(P=0.000), grading(P=0.000) and DM (P=0.015). The expression of MVD and VEGF was correlated with tumor staging and grading (P=0.000、P=0.001; P=0.000、P=0.039), but was not correlated with DM(P>0.05; P>0.05). The expression of GLUT1was positively correlated with HIF1a、MVD and VEGF in UCC (r=0.794, P=0.000; r=0.549, P=0.006; r=0.381, P=0.006).Conclusion:The expression of GLUT1was correlated with tumor grading, tumor staging, tumor hypoxic microenvironment and angiogenesis. And DM will increase the expression of GLUT1in tumor tissue in UCC.Part Ⅲ The interference effect of GLUT1shRNA for the bladder cancer cell line T24Objectives:By studying the effect of GLUT1on bladder cancer, To explore the role of GLUT1in progress of bladder cancer.Methods:Through in vitro studies using recombinant plasmid cytology technique to interference GLUT1level in bladder cancer cells, through Q-PCR、western blotting to detect the interference effect of RNA, through MTT, Flow cytometry and transwell to verify the effect of GLUT1for bladder tumor cell proliferation, apoptosis, cycle and invasion. By using2-deoxy-D-[3H]-glucose method to detect the changes in the rate of glucose uptake in cells.Results:By using A549tumor cells, we screened GLUT1-shRNA which the interference effect for the GLUT1was up to82%. The results of MTT, Flow cytometry and transwell shows that GLUT1-shRNA inhibited proliferation of T2458.69%(48h), promote apoptosis and necrosis of T24, the rate48h necrosis and apoptosis was47.41%and22.03%, inhibit the invasion ability of T24cells. GLUT1-shRNA reduces glucose uptake rate was62.39%in the T24cells.Conclusion:GLUT1express in human tumor cells, including human bladder cancer cells. Successfully constructed, screened pYr-1.1-GLUT1-shRNA Plasmid, the interference effects for the GLUT1was up to82%. In vitro GLUT1-shRNA can reduces glucose uptake in the T24bladder cancer cells, inhibition of proliferation, apoptosis, inhibition of invasion effect. GLUT1may play an important role in the progression of bladder cancer.Part Ⅳ Analysis of the polymorphisms of the GLUT1gene in urothelial cell carcinomas of the bladder and its correlation with P53, Ki67and GLUT1expressionsObjectives:The glucose transporter1(GLUT1) is a key rate-limiting factor in the transport and metabolism of glucose in tumor cells. Also recent studies have found that some single nucleotide polymorphisms (SNPs) of the GLUT1gene are associated with kinds of cancers’risk.Methods:Frequencies of two GLUT1SNPs (XbaⅠ G>T and HaeⅢ T>C) were investigated with PCR-RFLP method in314patients with urothelial cell carcinomas of the bladder (UCC) and204normal persons. And the expression of the P53, Ki67and GLUT1were assayed in51paraffin-embedded specimens of UCC patients by immunohistochemistry.Results:For the GLUT1XbaI G>T polymorphism, there was a decrease in the frequency of the TT genotype in the patient population versus the controls (38%vs.53%, P=0.033). This corresponded to an increase in the GT genotype in the patient population versus the controls (58%vs.43%, P=0.034). The T allele was decreased in frequency in the patient population versus the controls (64%vs.74%, P=0.022). No significant difference was seen between the patient population and the control population when the GG genotype was assessed. For the GLUT1HaeⅢ T> C polymorphism, there was, a decrease in the frequency of the CC genotype in the patient population versus the controls (4%vs.19%, P<0.001). There was an increase in the frequency of the TC genotype in the patient population versus the controls (83%vs.73%, P=0.006). There was a decrease in the frequency of the C allele in the patient population versus the controls (45%vs.56%, P=0.001). No significant difference was seen between the patient population and the control population when the TT genotype was assessed. The GLUT1Xba1genotype GG was more frequent in patients with higher tumor stage (P=0.014) and higher tumor grade (P=0.039). The GG genotype of the Xba1G>T SNP was significantly associated with higher Remmele immunoreactive score (IRS) of Ki67(P=0.031) and higher IRS of GLUT1(P=0.009). The Xba1G>T SNP showed no association with IRS of P53(P=0.935). The HaeⅢ T>C SNP showed no association with IRS of P53(P=0.964), IRS of ki67(P=0.145), IRS of GLUT1(P=0.167).Conclusion:The TT genotype of XbaⅠ G>T SNP and CC genotype of HaeⅢ T> C SNP may have protective effect in the carcinogenesis process of UCC. The GG genotype of the XbaⅠ G>T SNP was positively associated with tumor proliferation, glucose metabolism, tumor grade and stage. Therefore, the GLUT1might become a possible proliferation-related prognostic factor for UCC.更多还原