【作者】 李纲；

【导师】 张元芳；

【作者基本信息】 复旦大学 ， 泌尿外科学， 2003， 博士

【摘要】 背景与目的 膀胱移行细胞癌是最常见的泌尿系恶性肿瘤,近年发病率明显上升。该肿瘤大部分是浅表性的,常用经尿道电切的方法治疗,但一旦出现肌层浸润,不管用何种方法治疗,治疗效果不理想,预后差。目前还没有发现能早期发现或预测该肿瘤肌层浸润的标记物。大量研究表明血管生成是实体肿瘤生长、浸润进展和转移的必要条件,而血管生成因子的活性增高在肿瘤血管生成过程中起着十分重要的作用。近年发现血小板衍化内皮细胞生长因子(PD-ECGF/TP)在多种恶性肿瘤的生长、浸润进展和转移过程中起着重要作用。另一方面PD-ECGF/TP又有酶活性,具有胸苷磷酸化酶(TP)的作用,是体内代谢活化去氧氟尿苷和5-氟尿嘧啶的重要酶,PD-ECGF/TP的酶活性增高在胃、结肠和乳腺癌等多种恶性肿瘤中证实能增加去氧氟尿苷和5-氟尿嘧啶的抗癌作用。 本课题用逆转录-聚合酶链反应(RT-PCR)和Western印迹方法研究了?干扰素对膀胱癌RT-4和T24细胞PD-ECGF/TPmRNA和蛋白质表达的影响,及这种影响与去氧氟尿苷和5-氟尿嘧啶对膀胱癌RT-4和T24细胞的毒性作用之关系,同时用RT-PCR和免疫组织化学染色法研究了膀胱移行细胞癌组织中PD-ECGF/TP的mRNA和蛋白质的表达情况,探讨PD-ECGF/TP的mRNA和蛋白质的表达与膀胱移行细胞癌的浸润与复发等生物学行为的关系。第1部分 ?干扰素诱导的PD-ECGF/TP增强膀胱癌T24和RT-4细胞对去氧氟尿苷和5-氟尿嘧啶敏感性材料与方法 分别用RT-PCR和Western印迹检测高分化和低分化膀胱移行细胞癌的代表细胞株RT-4和T24细胞中PD-ECGF/TP mRNA和蛋白质的表达,MTS法确定去氧氟尿苷、5-氟尿嘧啶和丝裂霉素C对膀胱癌RT-4和T24细胞的细胞毒性作用。结果 未经?干扰素处理的RT-4和T24膀胱癌细胞,PD-ECGF/TP mRNA表达很低,?干扰素处理的RT??和T24膀胱癌细胞的PD-ECGF/TP mRNA表达明显增强,与浓度呈正相关,?干扰素作用4 h即见到PD-ECGF/TP mRNA表达增加,<WP=6>16 h达峰;Western 印迹检测到PD-ECGF/TP蛋白质表达呈现出与 mRNA表达相同的剂量依存性增强。?干扰素10000 U/ml单独时与RT-4或T24膀胱癌细胞作用48 h未见明显细胞毒性作用,经50 U/ml干扰素预处理24 h的RT-4和T24膀胱癌细胞对去氧氟尿苷和5-氟尿嘧啶的敏感性都明显增加。在RT-4细胞中,去氧氟尿苷的IC50从(9.7±0.2) mmol/L降至(3.3±0.9)mmol/L ( t = 8.64,P = 0.001);5-氟尿嘧啶的IC50从(130.0±21.2)?mol/L降至(49.3±18.4)?mol/L (t = 3.52,P = 0.02),丝裂霉素C的IC50在对照组为(1.5± 0.5)?mol/L,?干扰素预处理组为(1.4±0.3)?mol/L, 差异无显著性意义(t = 0.30,P = 0.78);在T24细胞中,去氧氟尿苷的IC50从(16.0±3.6)mmol/L降至(6.2±0.9)mmol/L (t = 2.82,P = 0.048),5-氟尿嘧啶的IC50从(826.7±155.1)?mol/L降至(346.7±38.9)?mol/L (t = 3.68,P = 0.021),丝裂霉素C的IC50在对照组为(2.0± 0.3)?mol/L,?干扰素预处理组为(2.3±0.6)?mol/L,差异无显著性意义(t = -0.58,P = 0.59)。第2部分 膀胱移行细胞癌中PD-ECGF/TP mRNA表达的研究 材料与方法 有明确病理学诊断的35例膀胱移行细胞癌, 其中Ta期14例,T1期12例,T2-4期9例; WHO分级:G1级 4例, G2级14例, G3级17例, 另有6例正常膀胱粘膜取自非肿瘤患者。膀胱癌组织的总RNA抽提按照ISOGEN试剂盒说明进行,取 2 ?g总RNA逆转录酶合成cDNA, 5倍稀释后取 2 ?l进行PCR, 所有标本用HGPRT mRNA作内对照, 目的PCR条带用NIH Image1.62软件根据面积与吸光度定量分析,每一标本的PD-ECGF/TP或VEGF mRNA表达用HGPRT标准化, 结果表示为PD-ECGF/TP mRNA 或VEGF mRNA与HGPRT mRNA的比值。结果 膀胱癌中PD-ECGF/TP mRNA表达水平是正常膀胱粘膜的3.86倍( t = 2.36, P = 0.024),其中Ta、T1和T2-4期膀胱癌分别是正常膀胱粘膜的1.33倍( t =1.18, P = 0.130)、4.02倍( t = 6.15, P < 0.001)和7.59倍( t = 5.84, P < 0.001)。PD-ECGF/TP mRNA表达在G1、G2和G3级膀胱癌中分别是正常膀胱粘膜的0.97倍( t = 0.11, P = 0.460)、2.80倍( t = 2.74, P = 0.013)和5.42倍( t =3.26, P = 0.004),说明膀胱癌的分级越高,PD-ECGF/TP mRNA表达越明显,低分级(G1级)肿瘤中的PD-ECGF/TP mRNA表达水平与正常膀胱粘膜无明显差别。膀胱癌中VEGF mRNA 表达明显高于正常膀胱粘膜,是正常膀胱粘膜的5.42倍( t = 6.14, P < 0.001), 与PD-ECGF/TP mRNA表达不同的是Ta、T1期膀胱癌和T2-4期膀胱癌都<WP=7>明显高于正常膀胱粘膜,其中Ta、T1和T2-4期膀胱癌的VEGF mRNA表达分别是正常膀胱粘膜的4.96倍( t =5.36, P < 0.001)、5.69倍( t = 6.63, P < 0.001)和5.85倍( t = 6.12, P < 0.001)。VEGF mRNA表达在G1、G2和G3级膀胱癌中分别是正常膀胱粘膜的3.12倍( t = 3.01, P = 0.017)、5.90倍( t = 7.11, P < 0.001)和5.61倍( t = 7.24, P < 0.001)。第3部分 膀胱移行细胞癌中PD-ECGF/TP的表达的免疫组织化学研究材料与方法46例膀胱移行细胞癌患者均有明确病理学诊断,采用TNM分期,其中Ta期12例,T1期11例,T2期6例,T3期11例,T4期6例,WHO病理分级为G1级3例,G2级23例,G3级20例。8例正常膀胱粘膜取自非肿瘤患者。免疫组织化学染色方法按照?更多还原

【Abstract】 Bladder transitional cell cancer is the most common malignancy in urinary system, and its morbidity has increased during these years. Superficial tumors comprises of more than two thirds of bladder transitional cell cancers and can usually be treated with transurethal resection. Treatment difficulty and poor prognosis ensued once muscle invasive tumors occurred. There has not yet been any markers which can be used to early detect the occurrence or the tendency of muscle invasive disease in bladder transitional cell cancer. Large quantities of researches indicated that angiogenesis is essential for growth, invasion and metastasis of solid tumors. The increased activity of vascular growth factors plays important roles in tumor angiogenesis. It has been rescently demonstrated that platelet-derived endothelial cell growth factor (PD-ECGF) plays an important role in the growth, invasion and metastatic process of several maligant tumor. The enzyme activity of PD-ECGF, which is identical to thymidine phosphorylase (TP), is an essential enzyme to activate 5’-deoxy-5-fluorouridine (5’DFUR) and 5-fluorouracil (5FU) in vivo. The anticancer activity of 5’DFUR and 5FU have been demonstrated increased by enzyme activity of PD-ECGF/TP in gastric, colon and breast cancer cells.This research investigated regulation effect of interferon gamma on PD-ECGF/TP mRNA and protein expression in RT-4 and T24 bladder transitional cancer cells using RT-PCR and Western blot, respectively. The relationship between these regulation effects and in vitro cytotoxicity of 5’DFUR or 5FU in RT-4 and T24 cells was also analyzed. The PD-ECGF/TP mRNA and protein expressions in resected bladder transitional carcinoma tissues were determined by RT-PCR and immunohistochemistry, repectively. The relationship between PD-ECGF/TP mRNA and protein expressions and biological behaviors of bladder cancer such as tumor invasion, grade and recurrence was also discussed.Section one Interferon gamma-induced up-regulation of PD-ECGF/TP enhances the cytotoxicity of 5’DFUR and 5FU in RT-4 and T24 bladder transitional cancer cells. <WP=10>Materials and MethodsRT-4 and T24 bladder cancer cells represents low-grade and high-grade bladder transitional cancer cells, respectively. RT-PCR and Western blot were used to analyze the mRNA and protein expressions in these cells. MTS method was performed to determine the cytotoxicity of 5’DFUR and 5FU and mitomysin C (MMC).Results Interferon gamma –untreated RT-4 or T24 bladder cancer cells expressed low level of PD-ECGF/TP mRNA. Markedly increased expression of PD-ECGF/TP mRNA was shown in RT-4 and T24 cells in a positive dose-related manner after interferon gamma treatment. The PD-ECGF/TP mRNA started to increase after 4 hours of interferon gamma treatment and reached the highest after 16 hours. Protein expression of PD-ECGF/TP as detected by Western blot showed increased expression in a dose-dependent manner similar to mRNA expression. No statistically significant cytotoxicity of interferon gamma only was shown at a dose up to 10000 U/ml even after 48 hours of treatment in both RT-4 and T24 cells. Interferon gamma pre-treatment at 50 U/ml for 24 hours significantly enhanced the sensitivity of RT-4 and T24 bladder cancer cells to 5’DFUR and 5FU. Interferon gamma reduced the IC50 of 5’DFUR from (9.7±0.2)mmol/L to (3.3±0.9)mmol/L (t = 8.64, P = 0.001) and IC50 of 5FU from (130.0±3.6)?mol/L to (49.3±18.4)?mol/L (t = 3.52, P = 0.02) in RT-4 cells. IC50 of 5’DFUR decreased from (16.0±3.9)mmol/L to (6.2±0.9)mmol/L (t = 2.82, P = 0.048) and from (826.7±155.1)?mol/L to (346.7±38.9)?mol/L (t = 3.68,P = 0.021) of 5FU after interferon gamma pre-treatment in T24 cells. IC50 of MMC, which is not activated by PD-ECGF/TP, was not altered by interferon gamma.Section two PD-ECGF/TP mRNA expression in bladder transitional cell cancerMaterials and MethodsA total of 35 cases with pathologically diagnosed bladder transitional cell cancer <WP=11>were included in this study, including 14 Ta, 12更多还原