聚合酶链反应对胆囊癌组织中细菌DNA片段的检测研究

目的　研究胆囊癌组织及相应胆汁中的细菌菌谱,以探讨胆囊癌的发生与细菌感染的关系。方法　采用聚合酶链反应（ＰＣＲ） ,对３７ 例胆囊癌组织标本及９ 例相应胆汁中的细菌ＤＮＡ 片段进行分子生物学扩增。结果　３７ 例胆囊癌组织标本中细菌ＤＮＡ 片段检出率为７８－３７％ （２９／３７）,９ 例相应胆汁中细菌ＤＮＡ检出率为７７－８％ （７／９）。结论　胆囊粘膜癌变可能与厌氧菌尤其是产气荚膜梭菌感染存在联系,厌氧菌与需氧菌协同作用,长期刺激胆囊粘膜可能是引起癌变的主要因素。     

PCR检测尿结核杆菌的临床应用价值

评价聚合酶链反应（ＰＣＲ）诊断泌尿系结核的临床应用价值，采用ＰＣＲ检测经病理证实为泌尿系结核９例，并报告１７例尿ＰＣＲ阳性的可疑肾结核抗痨治疗前后的临床情况，结果：９例泌尿系结核者，ＰＣＲ检测阳性７例；１７例尿ＰＣＲ阳性的可凝肾结核者经抗痨治疗３个月后，有１４例症状完全消失，尿常规转为正常，认为ＰＣＲ是临床快速诊断泌尿系结核的有效方法。     

应用聚合酶链反应检测生殖道溶脲脲原体的研究

采用培养法和聚合酶链反应（ＰＣＲ）检测了溶脲脲原体（Ｕｕ）标准菌株纯培养物、模拟临床生殖道Ｕｕ感染样本和５２例不育症病人的精液样本。结果培养法阳性的样本ＰＣＲ亦为阳性，并比培养法高出２～３个稀释度。若以培养法作为标准，ＰＣＲ的敏感性为１００％，特异性为８８％，准确性为９０％，提示ＰＣＲ是一种特异、灵敏和快速检测Ｕｕ感染的诊断方法。     

Detection of<Emphasis Type="Italic"> Helicobacter pylori</Emphasis> in bladder biopsy specimens of patients with interstitial cystitis by polymerase chain reaction

The cause of interstitial cystitis (IC) is still unknown. Several features suggest that it may be an infectious disease and it has compelling similarities to chronic gastritis. The identification of Helicobacter pylori as the cause of chronic gastritis focused attention on this organism. Many studies have been done investigating the role of H. pylori in the etiology of IC. Previous studies mostly determined the presence of H.pylori with antibodies in the serum samples of IC patients, but these methods may lead to false positive or negative results. We therefore investigated the presence of H.pylori in bladder biopsy specimens by using polymerase chain reaction (PCR), which is accepted as the most sensitive and specific test for detecting this organism. A total of 32 patients with IC were enrolled into the study. The PCR assay was performed on cold cup bladder biopsies of IC patients. Both positive and negative controls were included in each set of PCR reactions. Gastric biopsy specimens of peptic ulcer patients with proven H. pylori infection were used as positive controls. Bladder biopsies of all IC patients were negative for H. pylori DNA. PCR showed the presence of H. pylori in the positive controls in each cycle demonstrating that the PCR assay was working properly. Thus, there is no evidence that IC is the result of H. pylori infection. This study does not negate the possibility that other infectious agents may play a role in the etiology of IC.     

Ras oncogene point mutations in bladder cancer resistant to cisplatin

Summary Bladder tumors respond to cisplatin-based chemotherapy in 68% of cases, but only 30% will have a durable response. Recent studies have suggested that ras point mutations may produce cisplatin drug resistance in cell lines. To determine the role of ras point mutations in human tumors resistant to cisplatin, we evaluated ten tumors exposed to cisplatin and eight untreated bladder tumors for ras point mutations. Using polymerase chain reaction DNA amplification and allele-specific oligohybridization, we found that only one of the ten treated tumors and none of the untreated tumors harbored a ras point mutation. We conclude that ras point mutations occur infrequently in untreated bladder tumors and do not appear to correlate with cisplatin drug resistance in vivo.     

PCR检测非细菌性前列腺炎沙眼衣原体的初步研究

对３０例非细菌性前列腺炎（ＮＢＰ）患者的前列腺按摩液（ＥＰＳ）沙眼衣原体（ＣＴ）进行了聚合酶链反应（ＰＣＲ）检测，并与经二乙氨基葡聚糖处理的ＨｅＬａ细菌培养法进行对比研究，结果发现，６例ＰＣＲ和细胞培养双阳性，２１例ＰＣＲ和细胞培养双阴性，３例两种检测结果不一致，包括２例ＰＣＲ阳性但培养阴性，１例培养阳性但ＰＣＲ阴性，将ＰＣＲ与细胞培养进行比较，ＰＣＲ的敏感性为８５．７％，特异性９１．３％，阳性预     

An ultrastructural study of human bladder cancer treated by photodynamic therapy

An electron microscopic study has been carried out biopsy material of transitional cell carcinoma of human bladder, taken before and after photodynamic therapy (PDT), from 14 patients. It has been demonstrated that bladder cancer is highly sensitive to PDT using haematoporphyrin derivative (HPD, made in China) and laser irradiation. It was found that vascular endothelium within the tumour tissue was very sensitive to PDT, showing distinct changes as early as immediately after completion of a 20-min irradiation. Twenty-four hours after PDT, almost all the capillaries examined were necrotic and broken down into small fragments. The cancer cells were less sensitive to PDT, being damaged later and less seriously than the blood vessels. It has been concluded that in PDT-treated tumours the vascular endothelium is damaged primarily while the cancer cells are destroyed, to a considerable extent, secondarily as the consequence of structural damage to capillaries and functional disturbance in the microcirculation. We would speculate that the main factor influencing the final response is the actual concentration of HPD in various types of cell in the tumours.     

多重荧光PCR方法对结直肠癌进行微卫星不稳定检测及其临床价值

目的探讨多重荧光PCR方法检测结直肠癌微卫星不稳定（MSI）及其临床意义。方法将2004-2005年间进行手术治疗的110例结直肠癌患者建立队列，以多重荧光PCR方法检测结直肠癌MSI，并对MSI和微卫星稳定（MSS）结直肠癌患者的临床病理特点进行比较。结果多重荧光PCR方法扩增出所有患者的5个微卫星序列。其中MSI．H10例（8．1％），MSI-L13例（11．8％），MSS为87例（79．1％）。共检测BAT．26变异9例（8．2％）、BAT．25变异11例（10．0％）、D2S123变异11例（10．0％）、D5S346变异6例（5．5％）和D17S250变异8例（7．3％）。MSI-L、MSI．H和MSS组结直肠癌患者年龄比较，差异有统计学意义（P〈0．05）；其他临床病理特点差异无统计学意义（P〉0．05）。结论多重荧光PCR方法检测MSI结果稳定，宜于临床应用；MSI和MSS结直肠癌患者临床病理特点比较未见差异。     

Expression of survivin in bladder cancer cell lines using quantitative real-time polymerase chain reaction

ObjectivePrevious studies have reported that survivin expression is significantly associated with various malignancies including bladder cancer. However, the relationship between the expression of survivin and the tumor stage and grade of bladder cancer still require further study.MethodsTo determine whether survivin plays a role in the differentiation of bladder cancer cells, we conducted a preliminary study to examine the expression of survivin in bladder cancer cell lines.ResultsIn this study, we observed that the gene expression fold changes of survivin ranged from 3.2 to 16.7 in various tumor grades (G1–G4) of bladder cancer cell lines, which were higher than that in normal human urothelial cell line. With the worse differentiation of bladder cancer cell lines, the gene expression fold changes of survivin increased significantly (3.2-fold in RT4, 5.8-fold in 5637, 6.6-fold in T24, and 16.7-fold in HT1197). In addition, we observed different genotypes among various cell lines (C/C in HUC4449, C/G in RT4, C/G in 5637, G/G in T24, and C/C in HT1197). The relationship between survivin ?31 C/G polymorphism and various bladder cancer cell lines was non-significant. However, the overexpression of survivin may be associated with aggressive features of bladder cancer.ConclusionOur findings suggest that survivin could be a potential therapeutic target through the inhibition of cell proliferation in bladder cancer.     

多聚酶链反应寡聚核苷酸探针反向杂交在HLAⅠ类抗原分型中的应用

目的比较多聚酶链反应寡聚核苷酸探针（PCR—SSOP）技术与血清学方法对HLAⅠ类抗原A、B分型的准确性及分型精度。方法选取25例曾行血清学HLA-A、B分型的肾移植受者血标本行PCR—SSOP反向杂交法HLA—A、B分型。结果25例PCR—SSOP法HLA—A抗原分型均成功，检出A位点等位基因总数46个，单一位点4例，杂合子21例；检出B位点等位基因总数47个，单一位点3例，杂合子22例。血清学方法检出A抗原单一位点7例，其中2例经PCR—SSOP证实存在第2个位点，血清学A位点总误差率为12％；B抗原单一位点6例，1例PCR-SSOP证实存在第2个位点血清学B位点总误差率为8％。结论PCR—SSOP反向杂交法能对HLA—Ⅰ类抗原行中等分辨度进行等位基因分型，操作简单，分辨度及灵敏度高，结果解读客观。     

Human papillomavirus sequences are not detectable by Southern blotting or general primer-mediated polymerase chain reaction in transitional cell tumours of the bladder

Summary A large series of transitional cell tumours has been screened for the presence of human papillomavirus (HPV) sequences using Southern blotting and general primer-mediated polymerase chain reaction (GP-PCR). The latter technique allows the detection of a broad spectrum of both sequenced and unsequenced HPV types using two primer pairs located in the highly conserved L1 and E1 regions of the HPV genome. No evidence for HPV infection was found in 100 transitional cell tumours, 6 cases of carcinoma in situ, 2 adenocarcinomas and a squamous carcinoma of the bladder and 3 cases of cystitis. Similarly, 12 bladder tumour cell lines were HPV-negative in these assays. Cervical carcinoma cell lines containing from 1–3 to 600 copies of the HPV genome were used as positive controls and were scored positive in all assays by both Southern blotting and GP-PCR. It is concluded that despite the close proximity of the urothelium to the genital mucosa and the resemblance of some bladder tumours to known HPV-induced lesions in other tissues, HPV infection is absent or very uncommon in bladder tumours.     

Demonstration of fibroblast growth factor receptor-I in human prostate by polymerase chain reaction and immunohistochemistry

The expression and localization of fibroblast growth factor receptor-1 were investigated in human prostatic tissues with or without benign hyperplasia. Using a polymerase chain reaction method, we were able to demonstrate that prostatic tissues with benign hyperplasia expressed a significantly higher level of fibroblast growth factor receptor-1 mRNA than normal prostatic tissues (P < 0.01 by Anova). Western blot analysis using an antiserum against the receptor gave 2 bands with molecular weights of about 140 kDa and 80 kDa; these correspond to the expected sizes of the long and secreted forms of the fibroblast growth factor receptor-1, respectively. An immunohistochemical study using the same antiserum further demonstrated that the immunoreactive staining occurred mainly in the basal cells of the glandular epithelium and occasionally in the stromal cells. These results suggest that fibroblast growth factors may influence, at least in part, the proliferation of the epithelial cells seen in benign hyperplasia of human prostate. © 1995 Wiley-Liss, Inc.     

肾癌患者外周血中survivin mRNA的表达

目的探讨肾透明细胞癌患者外周血中survivin mRNA的表达及其临床意义。方法用RT-PCR方法检测30例肾透明细胞癌患者、10例正常健康人外周血中survivin mRNA的表达。结果30例肾透明细胞癌患者中有23例外周血中survivin mRNA表达阳性，阳性率为76．7％，与正常健康人（0％）相比差异有统计学意义（P〈0．001）；survivin mRNA的表达与临床分期密切相关（P〈0．01）；随肿瘤组织分化程度的降低，survivin阳性率有增加的趋势，但差异无统计学意义（P〉0．05）。结论肾透明细胞癌患者外周血中survivin mRNA可作为肾透明细胞癌微转移的一个监测指标。     

Detection of disseminated tumor cells in the lymph nodes of colorectal cancer patients using a real-time polymerase chain reaction assay

Introduction Postoperative treatment for colorectal cancer depends on tumor stage as defined by the International Union Against Cancer (UICC). Adjuvant chemotherapy is not recommended in patients without lymph node involvement (UICC stages I and II). As many as 20–30% of these patients, however, will develop recurrence. Aims and objectives We conducted this study to determine the presence of disseminated tumor cells in the lymph nodes by quantitative real-time polymerase chain reaction (QRT-PCR) for cytokeratin 20 (CK20) in an attempt to provide supplementary information compared to histopathological findings. Materials and methods Using a standard QRT-PCR assay, we examined primary tumors and 391 lymph nodes from 31 patients with completely resected colorectal cancer. Results Of the 31 primary tumors, 29 were positive for CK20 by QRT-PCR. Discussion An examination of the lymph nodes from the 29 patients with CK20-positive primary tumors revealed that 35 (92.1% sensitivity) of the 38 histopathologically positive lymph nodes and 54 (16.7%) of the 324 histopathologically negative lymph nodes were positive by molecular analysis. CK20 expression was detected in 10 (100%) of 10 patients with a histopathologically positive lymph node status (pN1). In 9 (47.4%) of 19 patients with negative histopathological results (pN0), we detected a CK20 mRNA signal in at least one lymph node. Whereas eight patients with histopathologically negative lymph nodes could be upstaged on the basis of the molecular findings, no patient would be downstaged. Conclusion Our results suggest that QRT-PCR for CK20 is a useful tool for the quantitative detection of micrometastases in the regional lymph nodes. We introduce a standardized procedure that integrates a molecular diagnostic technique in the clinical staging.     

Upregulation of cell adhesion through delta Np63 silencing in human 5637 bladder cancer cells

Some researchs have demonstrated that the loss of delta Np63 is associated with aggressive phenotypes and poor prognosis. However, other research indicates that delta Np63 is considered to have oncogenic properties. Delta Np63 overexpression is often observed in association with the oncogenic growth of squamous cell carcinomas and bladder cancer. In this study, we investigated the oncogenic role of delta Np63 in regulating cell adhesion in transitional cell carcinoma of the bladder (TCCB). The cells were stably transfected with the delta Np63 short hairpin RNA (shRNA) plasmid. Immunocytochemistry was performed to determine the knockdown efficiency. Tumour cells were studied for their ability to adhere to vascular endothelial cells. Confocal microscopy was used to analyse the changes in cytoskeletal F-actin. F-actin expression was measured by flow cytometry. Cell invasion ability was assessed using transwell chambers. The delta Np63-silenced tumour cells were shown to adhere more tightly than controls to vascular endothelial cells (P<0.05). The content of F-actin in the delta Np63-silenced cells was enhanced (P<0.05). The Matrigel invasion assays showed that human 5637 bladder cancer cells had a lower degree of motility when transfected with pdelta Np63-shRNA (P<0.05). In conclusion, silencing of the delta Np63 expression can enhance the adhesiveness of 5637 cells by inducing F-actin cytoskeleton production, and it will possibly inhibit the TCCB invasion and metastasis.     

Induction of mycobacteremia by intravesical bacillus Calmette-Guerin instillation in an experimental animal model and detection with polymerase chain reaction

PURPOSE: The aim of this study was to detect mycobacteremia by polymerase chain reaction (PCR), induced by the instillation of bacillus Calmette-Guerin (BCG) to guinea pig bladder. We also investigated the peak time and the effect of the dose of BCG in injured and non-injured bladder. The sensitivities of routine culture and PCR were also compared. MATERIALS AND METHODS: Five different doses (0, 0.069, 0.69, 6.9 and 69 mg.) of BCG were instilled into 5 injured and 5 non-injured bladders. Blood samples were collected at 0, 5, 15, 30 and 60 minutes following instillation for routine culture and PCR for each dose. A total of 50 female guinea pigs were used. RESULTS: Three of 5 samples (60%) obtained 30 minutes after the instillation of 69 mg. BCG into injured bladders were PCR positive. Furthermore, 4 of 5 samples (80%) were PCR positive when samples were obtained at the 60th minute following instillation. All the other samples were negative for PCR and routine culture. All the routine tuberculosis culture results were negative, including those which were PCR positive. CONCLUSIONS: Mycobacteremia was detected only in injured bladders and with high doses of BCG. PCR is a highly sensitive and rapid diagnostic method for detection of mycobacteremia.     

利用逆转录聚合酶链反应及DNA印迹杂交技术检测乳腺癌腋窝淋巴结微转移

目的　评价检测原发性乳腺癌腋窝淋巴结微转移的方法。方法　用逆转录聚合酶链反应(RT-PCR)和Southern杂交方法,检测腋窝淋巴结中CK-19基因表达。同时与免疫组织化学(组化)方法比较其检测敏感性。结果　RT-PCR、Southern杂交及免疫组化方法的检测敏感性分别为1∶5×10