尿脱落细胞CD44基因变异表达产物对膀胱癌诊断价值的研究

应用逆转录－聚合酶链反应和Ｓｏｕｔｈｅｒｎ印迹杂交技术对３０例尿液标本脱落细胞ＣＤ４４基因含Ｖ６外显子的表达产物进行检测，并与尿细胞检查结果进行比较。结果显示：９０％的膀胱癌尿脱落细胞均检测到ＣＤ４４基因含Ｖ６外显子的拼接变异转录物的过量表达；     

尿液脱落细胞CD44基因拼接变异体对膀胱癌的诊断价值

作者应用逆转录－聚合酶链反应和Ｓｏｕｔｈｅｒｎ杂交技术对２０例膀胱癌和２０例非癌对照组尿液脱落细胞ＣＤ４４基因拼接变异体进行检测,并与尿细胞学检查结果进行比较。结果显示：９０％的膀胱癌患者尿液脱落细胞均检测到ＣＤ４４基因拼接变异体的过量表达,而２０例非癌对照标本均未检测到这种异常。本技术诊断膀胱癌的敏感性为９０％,较尿细胞学检查的６５％明显提高。提示尿液脱落细胞ＣＤ４４基因拼接变异体是一种很有前途的用于膀胱癌早期无创伤性诊断的新的分子标记物。     

CD44基因变异性表达与膀胱癌的关系

为探讨膀胱癌的发生与ＣＤ４４基因变异表达之间的相关性，作者采用逆转录－聚合酶链反应和印迹杂交技术对４２例膀胱组织标本（２０例膀胱癌及其１０例正常膀胱组织，８例良性前列腺增生患者的正常膀胱组织和４例膀胱炎性组织）中ＣＤ４４基因含Ｖ６外显子的变异表达产物进行检测。结果显示：所有膀胱癌组织标本中均检测到ＣＤ４４基因含Ｖ６外显子的异常拼接变异转录物的过量表达，而２２例非癌膀胱组织中均未检测到这种异常的拼接变异转录物，且２例转移性膀胱癌与１８例非转移癌组织中上述变异表达产物的量及表现形式有差别。提示ＣＤ４４基因的变异表达（异常拼接和过量表达）与膀胱癌的恶性表型和转移特性密切相关，这种变异表达产物有可能成为膀胱癌早期诊断和对其转移潜能进行早期检测的较为理想的分子标记物，值得进一步研究。     

CD44基因在肾细胞癌中的表达

为探讨ＣＤ４４基因在肾细胞癌中的表达形式，应用逆转录聚合酶链反应（ＲＴＰＣＲ）及Ｓｏｕｔｈｅｒｎ杂交等方法，检测ＣＤ４４基因在１５例肾癌及对应癌旁组织中的表达。结果：肾癌组织均表达标准型ＣＤ４４（ＣＤ４４ｓ），并有１１例（７３％）表达了变异体，其中５例（３３％）表达约１４００ｂｐ带型，６例（４０％）表达约７００ｂｐ带型。癌旁正常组织也表达ＣＤ４４ｓ，但未表达变异体ＣＤ４４（ＣＤ４４ｖ）。结果提示，大部分肾癌组织中存在变异体ＣＤ４４，主要表现形式为７００ｂｐ（Ｖ１０）和１４００ｂｐ。     

膀胱肿瘤抗原检测膀胱癌的临床意义

目的 探讨膀胱肿瘤抗原（ＢＴＡ）对膀胱癌早期诊断的意义。方法 以ＢＴＡ和尿脱落细胞（ＶＵＣ）对３６例膀胱癌进行对比性检测。结果 ＢＴＡ的敏感性为４４．４％，ＶＵＣ为１６．６％，两者有显著性差异（Ｐ〈０．０１）。在早期膀胱癌，ＢＴＡ的敏感性为ＶＵＣ的６倍，进展期是ＶＵＣ的２．５倍。ＢＴＡ的特异性为９０％。结论 ＢＴＡ是一种简便、快速、无创伤性的检测方法，敏感性明显高于ＶＵＣ，尤其是早期膀胱癌中有较高     

膀胱癌尿脱落细胞染色体畸变的研究及临床意义

为明确荧光原位杂交在检测膀胱癌患者尿液，膀胱冲洗液中脱落细胞间期核染色体数目畸变的临床应用价值，收集２２例膀胱癌患者的尿液，２１例膀胱冲洗液，分别做尿细胞学、流式细胞术及荧光原位杂交的检测。同时收集５例正常人，５例前列腺增生患者尿液及２例膀胱冲洗液标本作为对照。结果显示：膀胱癌患者尿液组中尿细胞学、流式细胞术、荧光原位杂交分析阳性率分别为２７．３％（６／２２），４５．５％（１０／２２）和５０．０％（１１／２２），三者联合阳性率达６８．２％。膀胱冲洗液中阳性率则分别为２０．０％（４／２０），４０．０％（８／２０），７１．４％（１５／２１）和７６．２％（１６／２１）。认为荧光原位杂交技术检测膀胱癌患者尿液、冲洗液脱落细胞染色体畸变，可作为膀胱癌诊断的一项重要方法，并可能在术后监测复发以及预后判断中具有重要的临床意义。     

膀胱癌组织及尿脱落细胞CD44v6表达的研究

采用免疫组织化学技术观察尿脱落细胞ＣＤ４４ｖ６表达情况,以分析其对膀胱癌诊断的意义并探讨ＣＤ４４ｖ６在膀胱癌组织表达与临床病理特征的关系。材料与方法１９９７年９月至１９９８年３月住院膀胱癌患者４１例,非癌对照者３７例。术前留取新鲜尿液,制成细胞涂片４...     

膀胱癌尿脱落细胞端粒酶活性检测及其临床意义

目的检测尿脱落细胞端粒酶活性并探讨其临床意义。方法应用改良的端粒重复序列扩增（ＴＲＡＰ）银染方法,分别对膀胱癌组织、正常膀胱组织,以及膀胱癌患者和非尿路上皮肿瘤患者的尿脱落细胞、膀胱冲洗液进行端粒酶活性检测。结果１２例正常膀胱组织均无端粒酶活性,４８例膀胱癌组织中４４例（９１．７％）端粒酶阳性。膀胱癌患者尿液及膀胱冲洗液中脱落细胞端粒酶阳性率分别为８３．３％（４０／４８）和８７．５％（４２／４８）。１２例分化良好（Ｇ１级）膀胱癌患者中,尿液和膀胱冲洗液中脱落细胞端粒酶阳性率分别为７５．０％（９／１２）和８３．３％（１０／１２）。结论尿脱落细胞端粒酶活性检测敏感性高,可用于膀胱癌的早期诊断和术后随访。     

从膀胱癌尿脱落细胞中提取RNA进行RT—CPR的研究

目的：建立从膀胱癌尿液脱落细胞中提取ＲＮＡ进行ＲＴ－ＰＣＲ研究的实验方法。方法：从２０例膀胱癌患者尿液脱落细胞中提取ＲＮＡ，采用ＲＴ－ＰＣＲ技术扩增癌细胞中ＣＤ４４基因Ｖ６－Ｖ１１显子序列。结果：１８例尿液标本均可被扩增出所设计的相应片段，并经Ｓｏｕｔｈｅｒｎ印迹杂交证实。结论：临床上采用此技术可从分子水平上对大批膀胱癌２的癌基因表达产物进行无创伤性检测；为深入研究膀胱癌的发病机制、生物学行为和探     

CD44在颅内转移瘤及脑胶质母细胞瘤中的表达研究

目的　研究 Ｃ Ｄ４４ 粘附分子在颅内转移瘤及脑胶质母细胞瘤中的表达情况及其与这些肿瘤发生侵袭、转移的关系。方法　用抗 Ｃ Ｄ４４ 正常型（ Ｃ Ｄ４４ｓ） 蛋白单克隆抗体及抗 Ｃ Ｄ４４ 变体型（ Ｃ Ｄ４４ｖ３１０） 蛋白多克隆抗体免疫组化染色检测１０ 例正常脑组织、２０ 例脑胶质母细胞瘤和２０ 例颅内转移瘤中 Ｃ Ｄ４４ 粘附分子的表达情况。结果　１０ 例正常脑组织中 Ｃ Ｄ４４ｓ 和 Ｃ Ｄ４４ｖ３１０ 表达均阴性;２０ 例脑胶质母细胞瘤 Ｃ Ｄ４４ｓ 阳性表达率为１００ ％ , Ｃ Ｄ４４ｖ３１０ 表达阴性;２０ 例颅内转移瘤 Ｃ Ｄ４４ｓ 表达阳性率为９０ ％ , Ｃ Ｄ４４ｖ３１０ 表达阳性率为７０ ％ 。 Ｃ Ｄ４４ｖ３１０ 在颅内转移瘤及脑胶质母细胞瘤的表达差异有显著性（ Ｐ＜ ０ ．０１） 。结论　脑胶质母细胞瘤中 Ｃ Ｄ４４ｓ 的表达可能与其脑内侵袭过程有关,无 Ｃ Ｄ４４ｖ 表达可能与其很少发生颅外转移有关; Ｃ Ｄ４４ｖ 可能在颅内转移瘤向颅内转移的过程中起重要作用,且有可能成为颅内转移瘤诊治的有用指标之一。     

从膀胱癌尿脱落细胞中提取RNA进行RT-PCR的研究

目的:建立从膀胱癌尿液脱落细胞中提取RNA进行RT-PCR研究的实验方法.方法:从20例膀胱癌患者尿液脱落细胞中提取RNA,采用RT-PCR技术扩增癌细胞中CD_(44)基因V_6～V_(11)外显子序列.结果:18例尿液标本均可被扩增出所设计的相应片段,并经Southern印迹杂交证实.结论:临床上采用此技术可从分子水平上对大批膀胱癌患者的癌基因表达产物进行无创伤性检测;为深入研究膀胱癌的发病机制、生物学行为和探讨简便、有效的无创伤性检测方法奠定了实验基础.     

六位点微卫星组合用于膀胱癌诊断的研究

目的　探讨 6个微卫星位点组合在膀胱肿瘤诊断中的意义。　方法　选择 10个微卫星位点 (D9S16 2、D9S171、IFNA、D9S176、D9S195、D14S5 1、ACTBP2、D14S2 6 7、D17S6 95、D2 1S12 4 5 ) ,应用PCR SSLP法检测 32例膀胱肿瘤患者的肿瘤组织、膀胱冲洗液沉渣微卫星改变 ,15例非膀胱肿瘤患者为对照组。　结果　 32例膀胱肿瘤组织中 ,微卫星改变阳性 30例 ,敏感性为 93.8% (30 / 32 ) ,对照组膀胱冲洗液沉渣微卫星改变均为阴性。微卫星改变与膀胱肿瘤分期、分级无相关性。选取微卫星改变发生率最高的D9S171、IFNA、D14S5 1、D17S6 95、D2 1S12 4 5、ACTBP2 6位点组合微卫星改变阳性率为 90 .6 % (2 9/ 32例 ) ,与 10个微卫星位点组合相比差异无显著性意义。　结论　此 6个微卫星位点组合检测膀胱肿瘤可能具有较高的敏感性和特异性。     

Expression of CD44V2 in transitional cell carcinoma of the urinary bladder and in urine

CD44 is the principal cell surface receptor for hyaluronate. Variant forms of the receptor, produced by alternative splicing, have been found to be associated with tumor progression in a variety of cancers. Based on investigations at the RNA level, it has recently been proposed that expression of CD44 variant V2 was present in urothelial cancer but not in normal urothelium. Since a distinctive marker for urothelial cancer would be extremely useful, frozen sections of normal urothelium and urothelial cancer were examined for expression of standard CD44 and CD44V2. Frozen sections of specimens of 35 patients with transitional cell carcinoma of the bladder, 16 specimens of normal bladder and 5 ureters were examined. Immunohistochemical staining was performed using a polyclonal antibody to CD44V2 (PAB CD44V2), a monoclonal antibody to CD44V2 (MAB CD44V2) and a monoclonal antibody to CD44S (MAB CD44S). CD44V2 and CD44S were also measured in lysates of urine sediments from 21 patients by enzyme-linked immunoabsorbent assay (ELISA). All investigated transitional cell carcinomas expressed CD44V2. There was no differentiation between invasive and noninvasive carcinoma. CD44V2 was also expressed in normal urothelium. Standard CD44 was expressed by the transitional cell carcinoma, normal urothelium, musculature and interstitial tissue. The amount of CD44V2 and CD44S in lysates of urine sediments is not correlated to diagnosis. In contrast to investigations at the RNA level, CD44V2 on the protein level seems not to be a distinctive marker for urothelial cancer. Therefore, CD44V2 will not be a useful diagnostic marker for detection of transitional cell carcinoma.     

尿脱落细胞微卫星改变分析在膀胱肿瘤诊断中的应用

目的 选择6个微卫星位点，了解其在膀胱肿瘤诊断中的敏感性和特异性。方法 以外周血白细胞作为自身正常对照，检测31例膀胱肿瘤患肿瘤组织、尿脱落细胞以及10例正常人尿脱落细胞中的微卫星改变情况。结果 应用所选6个位点，诊断膀胱肿瘤具有90．3％的敏感性和100％的特异性，显高于尿细胞学检查的敏感性(10．3％)。结论 尿脱落细胞微卫星改变分析是一种无创、敏感、特异的诊断膀胱肿瘤的分子学方法。     

The simultaneous use of telomerase, cytokeratin 20 and CD4 for bladder cancer detection in urine

OBJECTIVE: Because of the low sensitivity of urinary cytological diagnosis of urinary bladder carcinoma, new molecular diagnostic methods have been proposed. We decided to verify the expression of telomerase mRNA coding for the catalytic component (hTRT), cytokeratin 20 (CK20) and CD4 antigen mRNAs in urine as possible diagnostic tool. METHODS: Evaluation of hTRT, CK20, CD4 mRNAs was performed in 50 ml of naturally voided urine of 205 patients of which 153 with bladder cancer (Tis, n = 11; TaGx, n = 4; TaG1, n = 25; TaG2, n = 26; TaG3, n = 8; T1G1, n = 16; T1G2, n = 17; T1G3, n = 20; T2G2, n = 6; T2G3, n = 13; T3G3, n = 7) and 52 controls. A quantitative expression of hTRT at mRNA level versus TRAP (telomeric repeat amplification protocol) assay was performed in 20 patients and 14 controls. The expression of RT-PCR for hTRT, CK20, CD4 versus urinary cytology was analysed in 44 patients with bladder cancer. Evaluating the three molecular markers together, the result was considered correct when at least two of the markers were positive, suspected when only one marker was positive and negative for diagnosis of tumour when all markers were negative. The performance of the diagnostic model resulted from the logistic analysis evaluated with receiver operating characteristics (ROC) curve analysis. RESULTS: The sensitivity detected for each tumour marker was as follows: for hTRT 90.8%, for CK20 84.3% and for CD4 was 64.7%, while the specificity was 94.2% for CD4 and 78.8% for both hTRT and CK20. When a simultaneous evaluation of the three tumour markers was considered, 88.2% of the diagnoses were correct, 11.8% were suspected for tumour and none were mistaken. When compared with cytology, the simultaneous use of the three markers allowed reaching a correct diagnosis in 88% of the cases in comparison to 25% by urinary cytology. The sensitivity in the detection of bladder cancer was higher for hTRT at mRNA level in comparison with the enzymatic activity detection with TRAP (90% vs. 35%) while the specificity for both markers resulted very high (100%). CONCLUSIONS: These data show that in the future the diagnostic improvement of urine based molecular markers for the detection of bladder cancer in the urine could improve the sensitivity of urinary cytology reducing the need of a cystoscopy.     

膀胱癌患者尿脱落细胞中细胞角蛋白20的表达及临床意义

目的　探讨膀胱癌患者尿脱落细胞中细胞角蛋白 2 0 (CK2 0 )的表达及临床意义。　方法　采用RT PCR方法检测 4 1例膀胱癌、2 0例其他泌尿系疾病患者及 10例正常人尿脱落细胞中CK2 0的表达。　结果　 10例正常人的尿脱落细胞均未表达CK2 0 ;2 0例非膀胱癌者中 3例表达CK2 0 ;4 1例膀胱癌患者中 37例表达CK2 0 ,三组间比较差别有显著性意义 (P <0 .0 1)。G1、G2 及G3膀胱癌的阳性率分别为 90 .2 % (2 3/2 5 )、88.8% (8/9)及 85 .7% (6 /7) ,三组比较差别无显著性意义(P >0 .0 5 )。　结论　CK2 0在膀胱癌患者尿脱落细胞中表达阳性率高 ,可用于膀胱癌的诊断及术后随访。