膀胱癌端粒酶活性的研究

目的 :探讨端粒酶活性和膀胱癌之间的关系。方法 :采用在PCR基础上建立的TRAP ELISA法对30例膀胱癌及 9例切缘组织和 7例正常膀胱组织中端粒酶活性进行半定量的研究。结果 :膀胱癌组总阳性率为83.3% (2 5 / 30 ) ,与切缘组 11.1% (1/ 9)和对照组 0 .0 % (0 / 7)相比差异有极显著性意义 (P <0 .0 1) ;后两者之间差异无显著性意义 (P >0 .0 5 )。端粒酶阳性表达率和表达强度与患者年龄、性别、病变部位、肿瘤大小、手术方式等无显著性相关 (P >0 .0 5 ) ,而端粒酶表达强度与细胞病理分级具有相关性 (P <0 .0 5 )。结论 :膀胱肿瘤的端粒长度和端粒酶活性对于判断疾病的恶性程度、预后、监测微小残瘤病灶和预示早期复发均有积极的意义     

羟喜树碱膀胱灌注对膀胱癌细胞端粒酶活性的影响

目的检测羟喜树碱膀胱灌注后膀胱癌细胞端粒酶活性变化,探讨其作用机理,从而指导临床用药。方法 50例膀胱移行细胞癌患者术前膀胱灌注羟喜树碱20mg,隔天1次,共3次。对术前术后标本采用端粒重复序列扩增法(TRAP-PCR-EIISA)检测肿瘤细胞端粒酶活性,并将其与患者临床特征及预后情况进行统计学分析。结果膀胱灌注前后肿瘤细胞端粒酶活性分别为12.69±3.71和3.92±1.07,差异有统计学意义(P0.05)。膀胱灌注前后端粒酶活性降低程度与患者年龄、性别、肿瘤数量、病理分级、发作次数无关(P0.05),与肿瘤直径、临床分期及预后情况有关(P0.05)。结论羟喜树碱膀胱灌注能抑制膀胱癌细胞端粒酶活性,具有明显的抗肿瘤作用。但其对浸润性癌及肿瘤体积大者效果差,且灌注后端粒酶活性变化小者预后差。     

Ha-ras mRNA与端粒酶在人膀胱肿瘤组织中的表达

目的　探讨人膀胱癌中Ha rasmRNA及端粒酶的表达与肿瘤临床指标的关系。方法采用原位杂交及银染端粒重复序扩增程序 (TRAP)反应的方法对 91例膀胱癌标本进行Ha rasmRNA及端粒酶活性的表达检测。结果　 91例膀胱癌标本中Ha rasmRNA与端粒酶活性检出率为 73 %与 92 % ,两者阳性率及阳性强度与肿瘤的恶性程度呈正相关 ,在不同临床分期中两者表达差异无显著性 (P >0 .0 5 )。结论　Ha ras癌基因与端粒酶是参与肿瘤的发生并影响肿瘤发展的重要因素 ,有望成为判断肿瘤恶性程度、自然病程及预后的评估指标。     

一氧化氮合成酶在膀胱癌中的表达及意义

目的　了解三种类型一氧化氮合成酶 (NOS)在膀胱癌中的表达情况 ,探讨其与肿瘤发生发展的关系。　方法　对 2 5例开放手术切除的膀胱癌组织标本进行三种NOS免疫组织化学染色 ,自动图像分析仪对染色程度分级 ;6例肾移植供体正常膀胱组织标本作为对照。　结果　诱导型NOS(iNOS)在肿瘤上皮细胞呈阳性表达 ,在正常膀胱移行细胞不表达或仅微弱表达 (P <0 .0 5)。内皮细胞型NOS (eNOS)在肿瘤基质毛细血管内皮细胞中的表达阳性率 1 0 0 % ,高于对照组中的 67%。两组标本中神经型NOS(nNOS)表达部位及强度相似。统计学分析显示iNOS和eNOS表达与肿瘤分级、分期无明显相关性 ,P均 >0 .0 5。　结论　iNOS及eNOS在膀胱癌的高表达可能与肿瘤的发生发展有关。     

端粒酶抑制剂与丝裂霉素C联合应用对膀胱癌的作用

目的:探讨端粒酶抑制剂对荷瘤小鼠肿瘤生长的影响及与化疗药物的协同作用。方法:应用端粒酶抑制剂齐夫多啶(AZT)联合化疗药物丝裂霉素C(MMC)治疗小鼠移植性膀胱癌(T24),观察其对抑瘤率、肿瘤端粒酶的表达及对肿瘤细胞凋亡的影响。结果:AZT、MMC、MMC加AZT的抑瘤率分别为12. 1%、29. 6%和43. 6%,AZT与MMC均能抑制肿瘤生长,并且AZT与MMC联用明显优于两者单独应用(P<0. 05 )。采用TUNEL法检测肿瘤细胞凋亡指数分别为(20. 23±0. 89)%、(8. 04±0. 12)%和(24. 09±1. 81)%。肿瘤端粒酶活性检测显示各组端粒酶阳性率分别为36. 5%、43. 6%和11. 8%,与对照组比较,AZT、MMC均有减少肿瘤端粒酶活性的作用(P<0. 05)。结论:MMC及AZT均能抑制小鼠膀胱癌T24细胞的生长及降低其端粒酶活性,诱导细胞凋亡,二者联用有相加作用。     

热休克蛋白70 mRNA在膀胱癌中的表达及与细胞凋亡的关系

目的　探讨热休克蛋白 70 (HSP 70 )mRNA在膀胱移行细胞癌中的表达及与细胞凋亡的关系。　方法　应用原位杂交法对 6 0例膀胱癌组织中HSP 70mRNA进行检测 ,原位DNA末端标记法 (TUNEL)检测细胞凋亡情况。　结果　 6 0例标本中 ,HSP 70mRNA阳性表达率为 5 6 .7% (34/ 6 0 )。HSP 70mRNA表达随肿瘤分级增高而增加 (P <0 .0 5 ) ;G1,G2 和G3 各级间两两比较 ,HSP 70mRNA表达差异也有显著性意义 (P <0 .0 5 )。随着肿瘤分期增高 ,HSP 70mRNA表达随之增加 (P <0 .0 5 ) ;Ⅰ ,Ⅱ和Ⅲ各期间两两比较 ,差异有显著性意义 (P <0 .0 5 )。肿瘤细胞凋亡指数 (AIs)随恶性程度的增高显著降低 (P <0 .0 5 ) ;HSP 70mRNA表达率和AIs呈负相关 (rs=- 0 .6 85 ,P <0 .0 5 )。　结论　HSP 70mRNA表达随膀胱癌恶性程度增加显著增高 ,细胞凋亡则显著降低 ,提示HSP 70表达能够抑制膀胱癌细胞的凋亡。     

胃癌与端粒酶活性表达及DNA倍体关系的研究

目的　揭示胃癌与端粒酶活性及DNA倍体的关系。方法　检测 30例胃癌标本 ,同时取无瘤残端作为对照。端粒酶检测采用端粒重复扩增 酶联免疫吸附法 (TRAP ELISA法 )。DNA倍体的测定采用流式细胞术 ,一步法检测DNA含量。结果　肿瘤瘤体端粒酶阳性率 83 3% (2 5 / 30 ) ,无瘤残端端粒酶阳性率 3 3%(1/ 30 ) (P <0 .0 5 ) ;端粒酶阳性瘤体平均直径 6 5cm ,阴性瘤体平均直径 3 6cm(P <0 .0 5 ) ;端粒酶阳性肿瘤淋巴转移率 5 3 3% (16 / 30 ) ,阴性者无淋巴转移 (0 / 5 ) (P <0 .0 5 ) ;端粒酶阳性肿瘤中异倍体肿瘤占 5 6 0 % (14/2 5 ) ,而端粒酶阴性者无异倍体出现 (P <0 .0 5 )。结论　胃癌端粒酶活性升高 ,端粒酶阳性肿瘤瘤体大 ,淋巴转移率高 ,且异倍体发生率高 ,预后差。提示端粒酶的激活与胃癌的发生发展有密切关系。     

端粒酶反义RNA基因转染抑制裸鼠人膀胱癌移植瘤生长的研究

目的　观察端粒酶反义RNA基因转染对裸鼠膀胱癌移植瘤细胞生长的影响。方法将脂质体介导的端粒酶反义RNA真核表达载体 (pBBS hTR)、空载体 ( pBBS 2 12 )及生理盐水注入移植瘤体内 (每天 3 0 0 μl,共 7d) ,应用端粒酶活性聚合酶链反应 酶联免疫吸附试验 (PCR ELISA)、苏木素 伊红 (HE)染色、透射电镜等连续观察 6周移植瘤组织细胞端粒酶活性和生长变化。结果　注射转染pBBS hTR组和空载体组瘤体积抑制率分别为 48.7%和 2 .8% (P <0 .0 5 )。转染pBBS hTR组端粒酶活性降低 ,细胞生长受抑制。电镜观察见典型凋亡特征。 结论　端粒酶反义RNA基因瘤体内注射转染有效地抑制裸鼠人膀胱癌细胞生长 ,具有潜在临床应用价值。     

端粒酶抑制剂与阿霉素联合应用治疗小鼠膀胱癌

目的 观察端粒酶抑制剂与化疗药物阿霉素联用抑制小鼠膀胱癌的协同作用.方法 AZT(端粒酶抑制剂齐夫多啶)4.5 mg、阿霉素0.1 mg、AZT 4.5 mg+阿霉素0.1 mg分组治疗T24膀胱癌荷瘤小鼠,观察肿瘤生长、细胞凋亡情况.结果 治疗后15 d,AZT、阿霉素、AZT+阿霉素各治疗组抑瘤率分别为35.6%、18.5%和43.2%.TUNEL法检测各组肿瘤细胞凋亡指数为(18.16±0.78)、(9.23±0.22)和(25.15±1.65).肿瘤端粒酶活性检测示各组端粒酶阳性率分别为24.5%、47.2%和12.3%,AZT、阿霉素均有减少肿瘤端粒酶活性的作用,且联用效果明显优于两者单独应用(P＜0.05).结论 AZT及阿霉素均能抑制小鼠膀胱癌T24细胞的生长及降低其端粒酶活性、诱导细胞凋亡,两者联用有相加作用.     

尿液端粒酶活性在膀胱癌诊断和监测复发中的意义

目的　探讨检测尿液中端粒酶活性在膀胱癌诊断和监测复发中的意义。　方法　采用PCR ELISA法检测 4 6例膀胱癌患者尿液中的端粒酶活性 ,并观察其中 2 0例术后尿液端粒酶活性变化及与复发的关系。　结果　膀胱癌患者术前尿液端粒酶活性 0 .6 0± 0 .5 6 ,明显高于对照组 0 .18± 0 .0 8,P <0 .0 0 1,肿瘤切除后恢复正常 ,肿瘤复发前再次上升 ;端粒酶活性随肿瘤恶性程度的增加逐渐升高 ;术前端粒酶活性与早期复发无关。　结论　尿液端粒酶活性检测在膀胱癌诊断中有参考价值 ,与膀胱癌恶性程度相关 ,可能成为监测肿瘤复发的重要辅助手段。     

卡介菌多糖核酸膀胱灌注对膀胱癌细胞端粒酶活性及细胞凋亡的影响

目的：探讨卡介菌多糖核酸（BCG—PSN）膀胱灌注对膀胱癌细胞端粒酶活性及细胞凋亡的影响。方法132例膀胱移行细胞癌患者术前膀胱灌注BCG—PSN20mg,隔天1次,共3次。对术前术后的标本采用端粒重复序列扩增法（TRAP—PCR—EI．ISA）和末端脱氧核苷酸转移酶介导缺口末端标记法（TUNEI。）分别检测肿瘤细胞端粒酶活性及细胞凋亡的情况。结果：膀胱灌注前后肿瘤细胞端粒酶活性分别为（12．78±3．67）和（3．89±1．12）,差异有统计学意义（P〈0．05）。灌注前后肿瘤细胞凋亡指数分别为（31．6±11．3）％和（69．1±12．4）％,差异有统计学意义（P〈0．05）。结论：BCG—PSN膀胱灌注能抑制膀胱癌细胞端粒酶活性,并诱导肿瘤细胞凋亡,具有明显的抗肿瘤作用。     

动脉化疗栓塞对膀胱癌组织中Ki-67和PCNA表达的影响

目的：探讨术前动脉化疗栓塞对膀胱癌组织Ki-67和增殖细胞核抗原（PCNA）表达的影响及其临床意义。方法：对30例膀胱癌患者化疗栓塞前后的肿瘤组织,应用免疫组织化学法测定Ki-67和PCNA的表达,并分析Ki-67和PCNA与膀胱癌病理分级和临床分期的关系。结果：对30例膀胱癌患者化疗栓塞前后的肿瘤组织,应用免疫组织化学法测定Ki-67和PCNA的表达,并分析Ki-67和PCNA与膀胱癌病理分级和临床分期的关系。结论：化疗栓塞前后Ki67中高度阳性表达率为70％、26.67％,PCNA为73.33％、20％,差异均有非常显著性意义（P〈0.01）,经随访24.6个月,复发率为16.67％。Ki-67和PCNA阳性表达及下降幅度与膀朊癌的病理分级、临床分期和患者术后复发率关系密切,两者阳性表达及表达强度呈正相关（P〈0.001）。结论：术前化疗栓塞能降低膀胱癌组织Ki-67和PCNA的表达,调节膀胱癌的分化程度,使肿瘤降级降期,减少术后转移,降低复发率,提高生存率。Ki-67和PCNA的表达可作为膀胱癌预后估计的指标。     

Telomerase activity Simplification of assay and detection in bladder tumor and urinary exfoliated cells

Detection of telomerase activity can differentiate malignant from benign cells. However, the original telomeric repeat amplification protocol (TRAP) methods had a number of limitations including a radioisotope labeling [α(32)P] dCTP [α(32)P] dGTP system. We developed digoxigenin labeled CX primer to detect telomerase activity without using radioisotope and attempted to detect telomerase activity of bladder tumor and exfoliated cells in bladder cancer patients. Telomerase activity was detected in 5 (71%) of 7 patients diagnosed with grade 1, 31 (97%) of 32 grade 2, and 11 (100%) of 11 grade 3 bladder tumors. In urinary exfoliated cells, 32 (82%) of 39 grades 1 or 2 bladder tumors were positive for telomerase activity but 20 (51%) of 39 were positive for urinary cytology (P < 0.01). Ten (91%) of 11 of grade 3 tumors were positive for telomerase activity and 11 (100%) of 11 were positive urinary cytology. Three of 100 noncancerous patients were positive for telomerase activity. Sensitivity, specificity, and positive predictive value of telomerase activity assay in urinary exfoliated cells were 84%, 97%, and 93%, respectively. Telomerase activity may be a useful diagnostic marker to detect the existence of immortal cancer cells in the urine.     

Telomerase activity is correlated with lower grade and lower stage bladder carcinomas

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that compensates for the progressive erosion of telomeres. The increasing interest in telomerase is motivated by the demonstration that most human carcinomas are telomerase positive. The potential use of telomerase activity in bladder carcinomas using a urine sample has been reported in several studies. However, little is known about the detection of telomerase activity in bladder carcinoma tissues. Herein, we investigate telomerase activity in bladder carcinoma tissues according to grade (G) and stage. MATERIAL AND METHODS: Telomerase activity was assayed by polymerase chain reaction enzyme-linked immunosorbent assay methods. Malignant lesions were assessed in 37 patients with bladder carcinoma and no malignant lesions were assessed in five patients with dysplasia or inflammatory bladder lesions. RESULTS: Twenty-three out of 37 carcinoma samples were telomerase-positive and one out of five control samples without carcinoma was telomerase-positive. The positive rates according to stage and grade were 83.3% for superficial and 42.1% for invasive stages and 83.3% for G1, 66.7% for G2 and 40.0% for G3. Telomerase activity was correlated with lower grade and lower stage bladder carcinomas. CONCLUSION: These results strongly suggest that reactivation of telomerase may differ between superficial and invasive bladder carcinomas and also between low grade and high grade bladder carcinomas.     

Nuclear cIAP1 overexpression is a tumor stage- and grade-independent predictor of poor prognosis in human bladder cancer patients

PurposeTo evaluate the tumor-related expression profile of cellular inhibitor of apoptosis protein 1 (cIAP1) and cellular inhibitor of apoptosis protein (cIAP2) in patients with bladder cell carcinoma (BCC) and to investigate its potential prognostic value.MethodsThe expression of cIAP1 and cIAP2 was examined immunohistochemically in archival bladder specimens from 32 normal controls and 102 consecutive patients who underwent surgical operations at our department from January 2004 through December 2005. Cytoplasm cIAP1 and cIAP2 expression was scored as 0 (negative), +1 (weak), +2 (medium), and +3 (strong). Nuclear cIAP1 expression was scored as 0 (0%), +1 (1%–25%), +2 (26%–50%), and +3 (>50%). Proliferation was determined by Ki67 staining as percentage of positive cells.ResultscIAP1 and cIAP2 expression were significantly increased in bladder cancer compared with normal bladder urothelium (cIAP1-C: P < 0.01, cIAP2-C: P = 0.017, cIAP1-N: P < 0.01). Nuclear staining of cIAP1 (cIAP1-N) was significantly associated with tumor stage (muscle invasive vs. non-muscle invasive, P = 0.03) and tumor grade (low vs. high, P = 0.01). Both the mean overall survival and mean recurrence-free survival were significantly decreased in the high cIAP1-N group compared to the low cIAP1-N group (low cIAP1-N: mean overall survival 62.7 months, high cIAP1-N: mean overall survival 45.6 months, P < 0.01; low cIAP1-N: mean recurrence-free survival 44.2 months, high cIAP1-N: mean recurrence-free survival 30.1 months, P < 0.01). cIAP1-N expression correlated strongly with KI67 expression (r = 0.744, P < 0.01).ConclusionNuclear cIAP-1 expression strongly correlated to bladder cancer stage, tumor grade, tumor recurrence and tumor related death. This marker expression was also appears to be a marker in bladder cancer prognosis.     

PCR-ELISA法检测膀胱肿瘤患者尿脱落细胞端粒酶活性

目的：探讨尿脱落细胞端粒酶活性变化在膀胱肿瘤诊断中的作用。方法应用PCR－ELISA法检测53例膀胱肿瘤患者尿液脱落细胞端粒酶的活性。结果：非膀胱肿瘤和膀胱肿瘤患者尿液脱落细胞端粒酶活性阳性率分别为64．15％（34．53）和7．69％（2／26）,健康对照者7例均为阴性,膀胱肿瘤患者与正常人及非膀胱肿瘤患者的端粒酶活性分别相比,差别均有极显著性意义（P＜0．001）。但端粒酶活性与肿瘤的分期分级无相关性。结论：尿脱落细胞端粒酶活性检测可以作为诊断膀胱肿瘤的无创性检测方法,但不能预测膀胱肿瘤的临床分期分级。     

环氧化酶-2基因在膀胱移行细胞癌组织中的表达及其意义

目的　探讨环氧化酶 2 (COX 2 )基因在膀胱移行细胞癌组织中的表达及其临床意义。方法　采用免疫组织化学酶标记链霉素亲和生物素法 (LSAB)对 78例膀胱移行细胞癌组织进行COX 2基因表达的检测。结果　在膀胱癌中COX 2主要呈胞核表达 ,染色阳性率为 5 6.41% ;COX 2表达与膀胱移行细胞癌组织分级、分期和预后呈高度正相关 (P <0 .0 1)。结论　膀胱移行细胞癌中COX 2异常表达与肿瘤分级、分期和疾病进展等生物学行为密切相关。     

Volume corrected mitotic index (M/V index) in human bladder cancer; relation to histological grade (WHO), clinical stage (UICC) and prognosis

A retrospective study was performed on 83 bladder cancer patients diagnosed at the Department of Surgery, Kuopio University Central Hospital, during the years 1965-1987. The follow-up time was 22 years, and the mean follow-up time of individual patients was 13 years (range 9.4-22 years). Histological grade (WHO), volume corrected mitotic index (M/V index) and clinical stage (UICC) were correlated to the survival of patients. Histological grade, M/V index and clinical stage were associated with crude survival (all causes of death included) with little predictive power. The recurrence of the disease could be predicted by the M/V index, but not by histological grade or clinical stage. When bladder cancer deaths only were included, histological grade (chi 2 = 26.6, p less than 0.001), M/V index (chi 2 = 6.6, p = 0.042) and clinical stage (chi 2 = 31.7, p less than 0.001) were clearly associated with prognosis. Also the metastasizing potential of bladder carcinomas could be predicted by the M/V index and by the histological grade at the time of primary diagnosis. Histological grade and M/V index were positively correlated (chi 2 = 16.7, p = 0.002, r = 0.47). In multivariate analysis clinical stage, histological grade and M/V index predicted prognosis in the order of importance.     

P_(53)蛋白和增殖细胞核抗原在膀胱癌中的表达及意义

采用免疫组化法对69例膀胱移行细胞癌中P_(53)蛋白及增殖细胞核抗原(PCNA)进行检测。发现膀胱癌中P_(53)蛋白的过度表达与病理分级无关,而与临床分期及预后有关。PCNA-LI与病理分极、临床分期及预后均相关,随着肿瘤分级、分期的增高,PCNA-LI呈明显上升趋势;P_(53)蛋白过度表达或PCNA高表达组术后5年生存率明显低于P_(53)蛋白非过度表达或PCNA低表达组。结果表明:P_(53)蛋白的过度表达在膀胱癌发生、发展中起着一定作用,PCNA是判定膀胱癌恶性程度及预后的重要指标,同时发现膀胱癌中P_(53)蛋白的过度表达与PCNA-LI相关。