基质金属蛋白酶血清含量与膀胱移行细胞癌浸润转移的关系

目的探讨基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的血清含量与膀胱移行细胞癌侵袭转移的关系。方法采用酶联免疫吸附试验(ELISA)对46例膀胱移行细胞癌患者血清中的MMP-2及MMP-9进行测定,并与18例正常血清进行对照分析。结果膀胱移行细胞癌患者同正常对照组比较,血清中MMP-2和MMP-9含量均显著增高(P<0.000),血清中MMP-2和MMP-9含量随着临床分期的升高均有显著性增高(P<0.0000),膀胱癌组织学Ⅱ、Ⅲ级患者血清中MMP-2和MMP-9含量明显高于I级(P<0.0000),但两者间比较无显著性差异(P>0.05)。结论MMPs与膀胱移行细胞癌的发生、发展及侵袭转移密切相关,MMPs血清学测定可作为判断膀胱肿瘤预后的指标之一。

人膀胱移行细胞癌染色体9p21.3-9p23杂合性缺失作图

目的 研究并确定人膀胱移行细胞癌染色体 9p2 1.3 -9p2 3区域杂合性缺失发生率和最小缺失区域 ,为寻找与克隆膀胱移行细胞癌相关的抑癌基因提供线索。方法 选取 7个微卫星多态性标记 (其中 6个位于 9p2 1.3 -9p2 3 ,另 1个位于 9q3 4作为对照 ) ,对 2 4例膀胱移行细胞癌组织及其对应的外周血淋巴细胞进行核素标记的聚合酶链反应和聚丙烯酰胺变性凝胶电泳。分析 9p2 1.3 -9p2 3区各微卫星位点杂合性缺失的发生情况及其与病理分期分级的关系。 结果 2 4例膀胱癌中的 2 0例 (83 .3 % )存在至少 1个微卫星位点的杂合性缺失。在 9p2 1.3 -9p2 3区域的 6个位点中 ,杂合性缺失率最高的为 9p2 3的D9S2 85 ,达66.7%(8/12 ) ;其次为 9p2 1的D9S1846,达 5 4.5 % (6/11)。位于 9q3 4.12的D9S182 1的杂合性缺失率为 5 4.5 % (6/11) ,而且在发生杂合性缺失的 6个病例中 ,有 5个被证明为部分染色体片段的缺失。结论 在 9p2 1.3 -9p2 3区域 ,可能至少存在 2个与膀胱移行细胞癌相关的候选抑癌基因 ,分别位于D 9S2

Laser Capture Microdissection Combined with RNA in vitro Linear Amplification Detecting the Relevant Genes of Bladder Cancer

Objective： Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma （BTCC）. However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determinc a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cclls from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods： Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results： That RNA integrity was good after LCM was confirmed by control experiment Ⅰ; By control experiment Ⅱ, the correlation between the number of LCM-shooting and RNA quantity undcr arranged conditions was preliminarily confirmed. About 0.5-2.5kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion： Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.

Clinical Significance of p53, Proliferating Cell Nuclear Antige and bcl-2 Expression in Bladder Transitional Cell Carcinoma

Objective: To correlate the frequency of p53 mutations, bcl-2 expression and the proliferation status (proliferating cell nuclear antigen, PCNA) in patients with bladder cancer with cell proliferation, apoptosis and their clinico-pathologic ?ndings. Methods: Para?n-embedded sections from 39 super?cial (T1G1-G3) and 23 invasive (T2-T4a G3 N0M0) primary transitional cell carcinomas (TCC) in the bladder were investigated immunohistochemically for p53, bcl-2 and PCNA. The median follow-up was 37 months; 24 had recurrences. The proliferation index (PI) was expressed as a percentage of the PCNA-positive cells in the tumor cells. Apoptosis was detected by terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of the TUNEL- positive tumor cells. Results: P53 mutation was identi?ed in 50 patients (80.6%). The mutation was most common in tumors of grade 3 (91.3%) as compared to grade 2 (78.5%) and grade 1 (72.7%, P<0.05). Stage pT2 tumors had a higher frequency of p53 mutation (95.7%) as compared to pTa-1 tumors (74.3%, P<0.01). Only 14 tumors (22.5%) expressed bcl-2; grade 3 tumors expressed bcl-2 signi?cantly more frequently (P<0.05); there was no correlation between bcl-2 and tumor stage. There was no interrelation between p53 mutation and bcl-2 expression (P>0.05). The PI ranged from 17.2% to 41.8% (median 22.4%) and the AI from 1.9% to 3.5% (median 2.9%) in bladder cancer. Statistical analyses revealed a close associations between PI, AI and tumor grade and stage of bladder cancer. Conclusion: P53 mutation correlates with invasion. P53 and PCNA overexpression may o?er valuable additional prognostic information in bladder tumors. With the progression of the tumor grade, cell proliferation may be accompanied by frequent apoptosis in bladder cancer, but the PI increased much more than the AI.

Effects of dendritic cell vaccines on hematogenous micrometastasis of bladder cancer carrying for PBL-SCID mice

Objective： To study the effect of dendritic cells loaded with whole tumor antigen on hematogenous micrometastasis of bladder cancer model in hu-PBL-SCID mice. Methods： T24-3 ceil subset was selected from human bladder transitional cell carcinoma T24 cell line by Boyden chamber system. The SCID mice intraperitoneally injected with 4 × 10^7 hu-PBL and subcutaneously injected with 3 × 10^6 T24-3 cells were named hu-PBL-T24-3-SCID model. Human IgG level in the blood plasma of mice was detected by ELISA, and human CD3^＋, CD4^＋, CD8^＋ T cells in blood and spleen cells of mice were detected by FCM analysis for human immune reconstruction study. Human CK20 mRNA expression in mice peripheral blood was detected by RT-PCR to investigate metastasis of tumor cells. The PBMCs were isolated from human peripheral blood, and were induced into DCs by co-culture with rhGM-CSF and rhlL-4 in vitro. The DC vaccines were produced by co-culturing with whole tumor antigen which was purified through freezing and melting T24-3 cell subset. After T24-3 cells injected into SCID mice for 5 weeks, the mice were treated with DC vaccines. Results： All mice were initially treated at 5th week. The expression of CK20 mRNA in peripheral blood of DC vaccines treated mice was the lowest. There was 2 mice showing CK20 mRNA expression and 3 mice with metastasis tumor in PBS group. MMP-7 mRNA expression in tumor tissues of DC vaccines treated mice was statistically lower than that of PBS group （P 〈 0.01）. Conclusion： DC vaccines have a good effect on hu-PBL-SCID mice bladder cancer model by reducing hematogenous micrometastasis.

Expressions and clinical significances of MMP-2 and TIMP-2 mRNA in bladder transitional cell carcinomas

Objective:The aim of the study was to investigate the expressions of matrix metalloproteinase-2(MMP-2) and tissue inhibitor of MMP-2(TIMP-2) mRNA in transitional cell carcinomas of bladder and discuss their clinical significances.Methods:Using RT-PCR and real time quantitative PCR(RQ-PCR) technique,the expressions of MMP-2 and TIMP-2 mRNA of 45 cases of bladder carcinoma(tumor group) and 10 cases of normal bladder tissue(control group) were analyzed.Results:MMP-2 and TIMP-2 were not expressed in control group.MMP-2 was expressed in 30 cases tumor samples and TIMP-2 was expressed in 26 cases.The expression of MMP-2 mRNA in non-muscle invasive bladder cancers and grade I cancers was lower than that in muscle invasive bladder cancers and grades II-III cancers respectively.The differences were statistically significant(P<0.05).The expression of MMP-2 in recurrent patients was higher than that in incipient patients.TIMP-2 mRNA expression decreased with grades and stage.The expression of TIMP-2 in non-muscle invasive bladder cancers and grade I cancers was higher than that in muscle invasive bladder cancers and grades II-III cancers respectively.There was statistical difference between two groups(P < 0.05).TIMP-2 expression in incipient patients was higher than that in recurrent patients,but the difference was not significant(P>0.05).Conclusion:These results suggest that MMP-2 and TIMP-2 play an important role in the invasion step of transitional cell carcinoma of bladder.MMP-2 may become a new approach to the diagnosis of bladder carcinoma.

Neoadjuvant Chemotherapy for Transitional Cell Carcinoma of the Bladder： A Single Centre Experience

Urothelial cancers usually recur distantly rather than loco-regionally. In patients with pT2 and pT3/pT4 tumors, local recurrence has been observed in 3-4% and 11-16%, respectively, whereas distant failure has occurred in 10-27% and 19-35%, respectively. Despite local therapy most patients with muscle invasive transitional cell carcinoma （TCC） of the bladder die of systemic relapse, indicating a need for effective adjunctive systemic treatment. We determined whether neoadjuvant chemotherapy improved overall survival. This study evaluated the role of neoadjuvant combination chemotherapy with gemcitabine/cisplatin （GC） in improving the outcome of this group of patients. A total of 44 patients （84% Male, 16% Female） with newly diagnosed bladder cancer （T3-4, N0-2, M0） were subjected to initial 3 cycles of GC, then managed according to response. Patients were assessed clinically after each cycle and by Interim CT scan after 3 cycles of chemotherapy and those who achieved complete or partial response underwent radical cystectomy. We enrolled 63 patients, 19 of whom were found to be ineligible; thus, 44 were assigned to receive neoadjuvant chemotherapy followed by surgery. Average size of the largest tumor was greater that 30mm in 77% patients. According to Computed Tomographic findings 70% patients belonged to Stage T4A. The overall response rate to GC was 50%, and incomplete response was achieved in 25% whereas 25% patients were lost to follow up. Twenty two patients who had complete response, underwent cystectomy and diversion. It was observed that those patients who underwent radical cystectomy with ureterosigmoidostomy had an increased serum creatinine in comparison to patients who had ileal conduit. The size of the effect is modest and combination chemotherapy can be administered safely without adverse outcomes resulting in delayed local therapy. Further efforts to identify the patients most likely to benefit from neoadjuvant therapy are necessary to optimize its use.

Prognostic value of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in bladder carcinoma

OBJECTIVES: To detect the level of matrix metalloproteinase-2 (MMP-2) mRNA and the tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA in bladder transitional cell carcinoma (BTCC), and to estimate the prognosis for bladder tumor based on the quality and quantity of MMP-2 and TIMP-2 mRNA. METHODS: Thirty-five samples of human BTCC and 15 normal fresh bladder tissues were studied by RT-PCR analysis followed by computer-assisted image analysis. RESULTS: The level of the MMP-2 mRNA in BTCC was significantly increased compared with that in normal bladder epithelium. The positive rates of MMP-2 and TIMP-2 mRNA were 71.4% and 65.7% in BTCC, and 66.7% and 60.0% in the normal bladder wall. The expression intensity of the MMP-2 mRNA by image analysis tended to increase with tumor grading and staging, which showed statistical significance. Similarly, the MMP-2 to TIMP-2 ratio also showed statistically significant difference between normal bladder tissue and bladder carcinoma (P