PCR-MASA检测胰腺癌十二指肠液中K-ras基因点突变

目的了解胰腺癌十二指肠液中K—ras基因点突变检测的临床价值。方法采用PCR-MASA(突变特异性等位基因扩增法)检测胰腺癌患者十二指肠液中K-ras基因点突变。结果胰腺癌患者十二指肠液标本中K—ras基因点突变率为17．4％(4／23),而被检测的急慢性胰腺炎、胰岛素瘤、壶腹癌、胆管癌、十二指肠乳头癌及胃癌病人十二指肠液标本均无K—ras基因突变。结论(1)PCR-MASA法简便、特异、敏感,扩增产物只需常规电泳、染色即可观察结果,无需酶切、杂交、放射性和非放射性显影。(2)对十二指肠液检测K—ras基因第12位密码子有无突变,可有助于判断胰腺病变良恶性及胰腺癌的诊断,但其实用价值尚有待进一步验证。     

病理性消化道息肉组织中端粒酶活性的检测及临床意义

目的探讨端粒酶在病理性消化道息肉中的检测及在早期胃癌或大肠癌诊断中的价值。方法端粒重复序列扩增法（TRAP—PCR—PAGE）检测175例胃镜活检标本端粒酶活性，标本均系电子内镜下取得的病理性胃、肠息肉组织。结果增生性息肉、腺瘤、炎性息肉和息肉样胃（肠）癌组织的端粒酶阳性率分别为6．2％、19．5％、15．4％和85．7％；同时随着息肉直径的增大、数目的增多，其端粒酶的阳性表达率也逐渐增高。结论端粒酶活性检测可作为消化道息肉恶变的早期预测指标。     

应用PCR和单克隆抗体方法从人粪便及肿瘤组织中检测大肠癌基因突变的研究

目的：检测大肠癌病人粪便中脱落细胞与肿瘤组织细胞的DNA，对比两者K—ras基因序列和突变位点的变化，为临床非介入性的、简便的大肠癌诊断提供理论和实验依据。方法：分离和提纯大肠癌组织及粪便中脱落细胞的DNA，用PCR(Polymerase chain reaction)法扩增K—ras基因，克隆K-ras基因PCR产物并进行DNA序列的测定。免疫组化检测大肠癌相应抗原的表达：采用ELISA法，用大肠癌单克隆抗体对大便样品进行检测。结果：同一病人的大肠癌组织细胞与粪便中的脱落细胞的K—ras基因的序列完全一致，并检测到1例K—ras基因点突变。K-ras基因突变与否与大肠癌病理类型和大肠癌单克隆抗体检测大便样品中脱落细胞的癌抗原表达水平有一定的相关性。结论：以大肠癌分子发病机理为基础的非介入性检测粪便中K-ras突变是可行的，有望成为正常人群大肠癌筛查以及诊断的新方法之一，可以为大肠癌的早期发现及监测大肠癌的发生、发展和预后提供理论和实验依据。     

胶质瘤中的多基因突变的分析

研究胶质瘤中癌基因c－myc的扩增、H－ras的突变及抑癌基因p53的5～8外显子的突变情况以及与胶质瘤的恶性程度的关系。采用差异性PCR（DPCR）及PCR－SSCP、PCR－RFLP等方法检测22例脑胶质瘤的基因突变情况。22例胶质瘤中c－myc扩增率为63．6％（14／22），H－ras的突变率为36．4％（8／22），p53的突变率45．5％（10／22）。结论：癌基因c－myc的扩增及抑癌基因p53的突变与胶质瘤的恶性程度有关（P＜0．05），而癌基因H－ras的突变则与胶质瘤的发生有关，与胶质瘤的恶性程度似乎无关（P＞0．05）。     

应用FISH技术检测宫颈脱落细胞中hTERC基医扩增的临床意义

目的：研究荧光原位杂交技术在检测宫颈脱落细胞中hTERC基因扩增情况,为女性子宫颈病变的筛查、早期诊断及预后提供新途径。方法：收集60例妇女患者进行宫颈脱落细胞的液基细胞学检查（TCT）、第2代杂交捕获法（HC2）高危型人乳头瘤病毒（HPV）检测（HPV—DNA）、FISH法hTERC基因检测及经阴道镜下宫颈活检病理证实,做出宫颈病变的最后诊断。结果：随着细胞学病变程度的加重及宫颈上皮内瘤变（CIN）级别增加,hTERC基因扩增阳性率增加（χ^2=11．111,χ^2=10．824,P〈0．05）;HPV感染率随着细胞学病变程度的加重而升高（χ^2=6．570,P〈0．05）;HPV阳性患者中hTERC扩增明显高于HPV阴性患者（χ^2=38．723,P〈0．05）。结论：hTERC基因扩增与宫颈细胞学和组织学密切相关,通过hTERC基因是否扩增可以很好区分低度与高度病变,hTERC基因的扩增可能是HPV感染致端粒酶活性增加的早期事件。     

内镜逆行胰胆管造影检查对胰腺癌的诊断价值

内镜逆行胰胆管造影检查(endoscopic retrograde cholangiopancreatography,ERCP)术日趋完善,已成为胰腺疾病诊断的重要手段,特别是对胰腺癌的诊断更有其他影像学检查不可比拟的优势.近年来随着内镜技术的发展,通过ERCP获取胰液、胰腺细胞学及组织学标本进行相关的肿瘤标志物及基因检测,可明显提高胰腺癌的早期诊断率.     

外周血K-ras基因突变和血清唾液酸联合检测在胰腺癌早期诊断中的意义

目的：研究外周血K-ras基因突变和血清唾液酸联合检测在胰腺癌早期诊断中的意义。方法：选择术前的36例胰腺癌胰腺良性病变患者,及20例正常人,同时行外周血K-ras基因突变和唾液酸血清水平的检测,利用统计学方法t检验和2χ检验进行数据分析。结果：胰腺癌中K-ras基因12密码子点突变率为69.4%,较胰腺良性病变的8.3%显著升高（P〈0.05）,外周血K-ras基因突变和血清唾液酸联合检测诊断胰腺癌的敏感性、特异性,比单独检测均有提高,且胰腺癌组和对照组之间差具有显著性意义（P〈0.05）。结论：提示该基因点突变检测对胰腺癌有诊断价值。K-ras基因点突变在某些良性病变中出现（如慢性胰腺炎）,这些良性病例突变列为胰腺癌发生的高危因素,对K-ras基因突变阳性者随访,有助于早期胰腺癌的发现。     

端粒酶及p53抑癌基因与原发性高血压的相关性研究

目的探讨原发性高血压患者外周血单核细胞的端粒酶活性与p53抑癌基因突变的相关性。方法用端粒酶TRAP—ELISA方法测定75例原发性高血压患者（高血压组）和50例健康者（对照组）外周血单核细胞端粒酶活性,同时用PCR-SSCP测定所有标本的053抑癌基因第5—8号外显子的突变情况。结果高血压级端料酶阳性率50．7％（38／75）,与对照组26．0％（13／50）相比,差异有统计学意义（P〈0．05）;与端粒酶活性有关的主要因素为年龄及收缩期血压,优势比分别为1．0418和1．0468。仅有2例高血压患者发生053抑癌基因的突变。结论端粒酶是细胞增殖活性的分子水平标记物,它的激活可能是原发性高血压的发病因素之一并能促进病变的发展。高血压病与053抑癌基因突变关系不明显,但该基因可能激活端粒酶活性。     

肝癌穿刺标本中端粒酶检测的临床研究

目的检测肝癌穿刺活检中的端粒酶活性，探讨其在肝癌诊断中的临床意义。方法采用TRAP．ELISA技术检测34例肝活检标本中的端粒酶活性。结果肝癌中端粒酶阳性80％，且明显高于其他检测指标。结论显示端粒酶检测是一项敏感性较高的指标，阳性率明显高于其他检测结果，配合细胞学检查，可提高肿瘤的早期诊断率，并有可能成为肝癌的生物学指标。     

PCR及限制性酶切技术检测急性髓细胞白血病H-ras基因点突变

为了解急性髓细胞白血病(AML)H-ras基因点突变的情况,应用聚合酶链反应(PCR)和限制性酶切图谱分析技术,对55例初诊AML患者进行了检测,发现16例(29.1％)存在H-ras基因点突变,此突变与AML的发生及其某些临床指标有一定关系。H—ras基因突变可望成为AML诊断分型、指导治疗及判断预后的一项有价值的指标。     

Clinical usefulness of KRAS mutational analysis in the diagnosis of pancreatic adenocarcinoma by means of endosonography-guided fine-needle aspiration biopsy

AIM: To establish the usefulness of KRAS mutational analysis in the diagnosis of pancreatic adenocarcinoma by comparing this technique with conventional cytology in aspirates obtained by endosonography-guided fine-needle aspiration. METHODS: All consecutive patients with pancreatic focal lesions undergoing endosonography-guided fine-needle aspiration were included. Samples were obtained with the concurrence of an attendant cytopathologist. Detection of codon-12 KRAS mutations was performed by the restriction fragment length polymorphism-polymerase chain reaction method. The effectiveness of conventional cytology, KRAS mutational analysis and their combination was established with respect to the definitive diagnosis. A cost-effectiveness analysis was also performed. RESULTS: Thirty-three patients had pancreatic adenocarcinoma and 24 patients had other lesions. A total of 136 samples was obtained. In patients in whom specimens were adequate (93% for cytology; 100% for mutational analysis), the specificity of both techniques was 100%, whereas the sensitivity favoured cytology (97% vs. 73%). When inadequate samples were considered as misdiagnosed, a combination of both techniques reached the highest overall accuracy (cytology, 91%; mutational analysis, 84%; combination of both, 98%). CONCLUSIONS: Cytology from aspirates obtained by endosonography-guided fine-needle aspiration is the most precise single technique for the diagnosis of pancreatic adenocarcinoma. However, when adequate specimens are not available to reach a cytological diagnosis, the addition of KRAS mutational analysis represents the best strategy.     

Identification of tobacco-derived compounds in human pancreatic juice

Cancer of the pancreas is the fourth leading cause of cancer mortality in the USA with an estimated 28 900 deaths in 2001. Several factors have been implicated in the etiology of this disease. However, at present, only cigarette smoking has been positively associated with pancreatic cancer. It is our working hypothesis that tobacco-derived compounds can be delivered to the pancreas where, upon metabolic activation, they can initiate carcinogenesis. Our current investigation was conducted to determine whether cotinine and tobacco-specific nitrosamines (TSNA) are present in human pancreatic juice. Smoking status was assessed by the determination of levels of urinary cotinine and was further supported by quantifying nicotine in hair. The TSNA were extracted from the pancreatic juice of 18 smokers and 9 nonsmokers by supercritical carbon dioxide that contained 10% methanol. The extracts were analyzed for TSNA, namely, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), by gas chromatography with mass spectrometric detection using a selected ion monitoring technique (GC-SIM-MS). Twenty-three extracts of human pancreatic juice were also analyzed for the presence of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by GC-SIM-MS and by gas chromatography interfaced wit a thermal energy analyzer (GC-TEA; TEA, a nitrosamine-specific detector). Cotinine was detected in all analyzed samples of pancreatic juice from smokers (129 +/- 150 ng/mL juice; mean +/- standard deviation) and was present in only two of the nine samples of pancreatic juice from nonsmokers. Its levels in these two samples were 7 and 9 ng/mL juice. NNK was detected in 15 of 18 samples (83%) from smokers at levels from 1.37 to 604 ng/mL pancreatic juice. In nine samples of pancreatic juice from nonsmokers, NNK ranged from not detected (in three samples) to 96.8 ng/mL juice. In pancreatic juice from smokers the mean level of NNK (88.7 +/- 161 ng/mL juice) was significantly higher (p < 0.04) than in that from nonsmokers (12.4 +/- 31.7 ng/mL juice). In addition to NNK, NNN was found in two samples of pancreatic juice of smokers at levels of 68.1 and 242 ng/mL juice; NNN was not detected in any other sample. NNAL was present in 8 of 14 pancreatic juice samples (57%) from smokers and in three of nine samples (33%) from nonsmokers. This research presents preliminary data that supports the hypothesis that pancreatic tissue is exposed to TSNA and that they may be important contributors to pancreatic carcinogenesis in humans.     

肺癌组织及支气管冲洗液中肺癌细胞的端粒酶活性分析

目的：研究纤支镜下取得的活检肺癌组织、支气管冲洗液中肺癌细胞的端粒酶活性,探讨并在肺癌诊断中的意义。方法：应用银染－TRAP,检测了26例癌组织、4例良性肺疾病组织,相应30例支气管冲洗液中的端粒酶活性表达。结果：26例肺癌组织中,22例端粒酶表达阳性,阳性率为84．64％。26例肺癌患者支气管冲洗液中,12例端粒酶表达阳性,阳性率46．15％。结论：端粒酶可能成为肺癌早期诊断的重要标志物,可能成为肿瘤治疗方面的重大突破口。     

端粒酶在前列腺癌组织中的活性表达及意义

目的探讨端粒酶在前列腺癌组织中的表达及其临床意义。方法采用免疫组织化学SP法检测41例前列腺癌组织、18例前列腺增生组织和11例正常前列腺组织中端粒酶活性的表达,并与肿瘤的临床分期、分级及淋巴结或骨转移情况进行比较分析。结果41例前列腺癌组织中湍粒酶的阳性表达率为82.9%,明显高于良性前列腺增生(5.6%)及正常前列腺组织,而且表达率随肿瘤的临床分期、分级升高而增加,有淋巴结或骨转移者表达率高。结论端粒酶活性在前列腺癌组织中表达率高,在浸润转移过程中起重要作用,可作为前列腺癌早期诊断及估计预后的指标之一。     

An anti-K-ras ribozyme suppresses oncogene expression and cell growth of human pancreatic cancer

Hammerhead ribozymes are effective modulators of gene expression due to their simple structure, site-specific cleavage activity and catalytic potential. The K-ras oncogene is thought to play an important role in the growth of pancreatic cancer, because an activated (mutated) ras gene is found in approximately 90% of human pancreatic cancers. In this study, we designed a hammerhead ribozyme directed against K-ras mRNA at codon 25 [K-ras Rz (25)], and investigated its efficacy in a cultured human pancreatic carcinoma cell line, MIA PaCa-2. K-ras Rz (25) significantly reduced the cellular K-ras mRNA level when introduced into the MIA PaCa-2 cells. The ribozyme suppressed cell growth. K-ras Rz (25) appears capable of reversing the malignant phenotype in human pancreatic carcinoma cells.     

液基细胞学技术在纤维支气管镜刷检细胞学检查中的应用

目的 探讨液基细胞学检测（LCT）技术在纤维支气管镜（纤支镜）刷检细胞学检查中的临床应用价值.方法 300例经纤支镜组织活检确诊为肺癌的患者,分别应用LCT技术与传统涂片方法进行细胞学检查对比,比较两者的阳性检出率及组织学分型的准确率.结果 300例肺癌患者,传统细胞学方法检出132例阳性（44.0%）,LCT技术检出201例阳性（67.0%）;各型肺癌中,LCT检测腺癌的组织学分型准确率明显提高（P〈0.05）.结论 LCT技术较传统涂片法的制片术有极大改进,在肺癌早期诊断及健康人群普查中有较高的应用价值.     

人胰腺癌细胞中端粒酶活性的检测

目的 观察银染的端粒重复序列扩增法（TRAP）和TRAP－ELISA两种方法检测人胰腺癌细胞株中端粒酶活性的特异性和敏感性。方法 用TRAP－银染法和TRAP－ELISA两种方法检测人胰腺癌细胞株及人皮肤成纤维细胞中端粒酶活性,并检测经RNase和加热处理的阴性对照标本。结果 10个以上的P3细胞均为端粒酶阳性,而RNase和加热处理的标本均为阴性,胰腺癌细胞株Patu-8801亦为阳性,人皮肤成纤维细胞为阴性。结论 两种方法均为特异、敏感的端粒酶活性检测法,TRAP－ELISA方法更简单、方便,并证实两株人胰腺癌细胞株均为端粒酶阳性。