人大肠癌细胞系HR 8348和HCe 8693 K－ras基因的点突变分析

本文采用修饰引物的聚合酶链式反应（ＰＣＲ）所建立的限制性片段长度多态性（ＲＦＬＰ），并结合ＰＣＲ直接测序方法，首次分析了我国建株大肠癌细胞系ＨＲ８３４８和ＨＣｅ８６９３的Ｋ－ｒａｓ基因点突变。发现该两细胞系均存在ｋ－ｒａｓ第１２密码子的点突变，其方式均为ＧＧＴ→ＧＡＴ；但未发现Ｋ－ｒａｓ第１３和６１密码子的突变。     

PCR—SSP快速检测胰腺癌Ki－ras基因点突变

ＰＣＲ—ＳＳＰ快速检测胰腺癌Ｋｉ－ｒａｓ基因点突变戴存才刘训良杜竞辉ＰＣＲ—ＳＳＰ快速检测胰腺癌Ｋｉ－ｒａｓ基因点突变@戴存才@刘训良@杜竞辉...     

PCR—RFLP检测胰腺癌石蜡组织中K—ras基因点突变

ＰＣＲ－ＲＦＬＰ检测胰腺癌石蜡组织中Ｋ－ｒａｓ基因点突变肖尚喜１曹立宇２张军１方有兵１＊本课题由安徽省卫生厅医学科研基金资助（９４－０４）作者单位：１安徽医科大学第一附属医院检验科，合肥２３００２２２安徽医科大学病理学教研室，合肥２３００３２作者简介...     

肿瘤坏死因子α与反义寡脱氧核苷酸联合作用对人胰腺癌细胞生长的影响

目的探讨肿瘤坏死因子α（ＴＮＦα）与反义寡脱氧核苷酸的联合应用对胰腺癌细胞生长的影响。方法用ＴＮＦα和硫代正磷酸修饰的反义寡脱氧核苷酸对本室自建的胰腺癌细胞系（ＰＣ－２）进行处理，并与单独用药和未用药组进行对比。用细胞计数和噻唑蓝（ＭＴＴ）法分析细胞生长速度和增殖率，用ＲＴ－ＰＣＲ－ＳｏｕｔｈｅｒｎＢｌｏｔ检测药物对靶基因表达的影响，并对细胞的凋亡状况进行原位检测。结果联合用药组较单独用药组使细胞生长速度减慢的作用明显加强；针对Ｋｉ－ｒａｓ和ｃ－ｍｙｃ帽区的反义硫代正磷酸修饰的寡脱氧核苷酸（简称反义Ｋｉ－ｒａｓ和反义ｃ－ｍｙｃ）可以明显抑制内源性靶基因Ｋｉ－ｒａｓ和ｃ－ｍｙｃ的表达；ＴＮＦα与反义Ｋｉ－ｒａｓ、反义ｃ－ｍｙｃ联合应用凋亡细胞发生率为８．０％和８．４％，高于其分别单独应用的５．２％、４．８％和５．４％。结论ＴＮＦα与反义寡脱氧核苷酸的联合应用对细胞生长的影响大于单独作用。     

膀胱癌组织中K－ras基因点突变及其基因产物P21的表达研究

为在分子生物学水平探讨膀胱癌的发生发展以及恶性程度的判别和预后评价的指标，作者应用聚合酶链反应（ＰＣＲ）结合单链构象多态性分析（ＳＳＣＰ）和免疫组织化学方法，研究了５４例膀胱癌组织和５例正常膀胱组织的Ｋ－ｒａｓ基因第１２位密码子点突变和其产物Ｐ２１的表达情况。结果表明：５４例膀胱癌组织中，仅有４例Ｋ－ｒａｓ基因第１２位密码子点突变和３４例Ｐ２１蛋白阳性表达，并且浅表性肿瘤Ｐ２１阳性表达率明显低于浸润性肿瘤。提示Ｋ－ｒａｓ基因点突变在膀胱癌中发生率较低，Ｐ２１蛋白的过量表达主要是膀胱癌晚期表现，它可能与Ｈ－ｒａｓ和Ｎ－ｒａｓ基因突变有关。为肿瘤病因学研究和临床研究提供了有用的资料。     

结肠粘膜,腺瘤和腺癌组织中Ki—ras癌基因突变的检测

对４１例石蜡包埋，同时含在可分割的正常粘膜，癌旁腺瘤和腺的手术切除结肠标本；１０例石蜡包埋，内窥镜摘除的腺瘤标本，分别用限制性片段长度多态性，单链构象多态性和Ｓ１核酸酶保护技术对Ｋｉ－ｒａｓ癌基因第１２和第６１密码子突变进行了检测，结果发现：正常粘膜中无Ｋｉ－ｒａｓ癌基因突变“腺癌组织中有５１％（２１／４１例），癌旁腺瘤中有２２％（９／４１例）非癌旁腺癌瘤中有１０％（１／１０例）有Ｋｉ－ｒａｓ癌基     

不同肺腺癌细胞系N—ras,P53基因突变核酸测序分析

应用聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎＰＣＲ）和ｄｓＤＮＡｃｙｃｌｅｓｅｑｕｅｎｃｉｎｇｓｙｓｔｅｍ技术对倍外培养的人肺腺癌细胞系ＬＴＥＰ－ａ２和ｈＬ中Ｎ－ｒａｓ癌基因及Ｐ５３抑癌基因外显子５、７进行核酸序列测定分析。结果表明Ｎ－ｒａｓ突变热点第１２、１３、６１密码子未见异常Ｐ５３抑癌基因第１５４密码子均发现ＧＧＣ－ＧＴＣ突变。经光盘检索（美国Ｓｉｌｖｅｒｐｌａｔｔｅｒ公     

ras基因点突变及其产物P21蛋白的表达在肺癌研究中…

ｒａｓ基因家族由Ｈ－ｒａｓ，Ｋ－ｒａｓ组成，其表达产物为Ｐ２１蛋白，ｒａｓ基因主要通过点突变激活，左突变位点是密码子１２、１３、和６１。肺癌中ｒａｓ基因点突变绝大多数发生在肺腺癌，与吸烟有一定的关系。主要是Ｄ－ｒａｓ基因第１２密码子的点突点。Ｇ－Ｆ突变常见，导致甘氨酸被缬氨酸所代替。有Ｋ－ｒａｓ基因点突变的肺腺癌分化差，患者预后也差。Ｐ２１蛋白在肺癌中的表达一般随着分化的升高而升高。Ｐ２１蛋白阳性     

不同肺腺癌细胞系N－ras、p53基因突变核酸测序分析

应用聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎＰＣＲ）和ｄｓＤＮＡｃｙｃｌｅｓｅｑｕｅｎｃｉｎｇｓｙｓｔｅｍ技术对体外培养的人肺腺癌细胞系ＬＴＥｐ－ａ＿２和ｈＬＡ中Ｎ－ｒａｓ癌基因及ｐ５３抑癌基因外显子５、７进行核酸序列测定分析。结果表明Ｎ－ｒａｓ突变热点第１２、１３、６１密码子未见异常。ｐ５３抑癌基因第１５４密码子均发现ＧＧＣ→ＧＴＣ突变（Ｇｌｙ→Ｖａｌ变异）。经光盘检索（美国Ｓｉｌｖｅｒｐｌａｔｔｅｒ公司提供的ＣＯ－ＲＯＭＭＥＤＩＬＩＮＥ）分析了１９８８～１９９３年间的专题文献，尚未见肺癌ｐ５３基因第１５４密码子突变的报道。     

中国人K-ras原癌基因外显子1序列分析

目的 为了解肺癌患者血液中Ｋ－ｒａｓ原癌基因突变情况。方法 采用聚合酶链式反应－测序方法对１２例肺癌患者及３１例正常人血细胞中Ｋ－ｒａｓ原癌基因外显子１进行测序分析。结果 所有４３例标本中Ｋ－ｒａｓ基因第１１位密码子均为ＣＣＴ（编码脯氨酸），和对侧链测定为ＡＧＧ，与国外测定的标准序列不同，考虑可能为人种变异，结论 中国人Ｋ－ｒａｓ原癌基因第１１位密码子ＣＣＴ，因该密码子与突变位点紧邻，这对临床基因     

Oncogene expression and point mutation in human pancreatic adenocarcinomas]

By nucleic acid hybridization and PCR techniques, oncogene expression in normal human pancreatic tissue as well as 4 human pancreatic adenocarcinoma cell lines were studied. Additionally, c-Ki-ras gene point mutation were also detected in the paraffin embedded sections collected from 34 cases of surgical specimens resected due to pancreatic adenocarcinoma. The results showed that none of the 5 oncogenes described was expressed in the normal human pancreatic tissue, whereas, all the human pancreatic adenocarcinoma cell lines expressed c-myc and c-Ki-ras oncogenes. 28 out of 34 cases (82.4%) of human pancreatic adenocarcinomas had c-Ki-ras gene codon 12 point mutation.     

Characteristics of plasma DNA and its application for detection of K-ras gene mutation

DNA diagnosis is useful and significant for clinical oncology, but its use is limited due to a difficulty in preparing tumor-derived DNA materials. To overcome this problem, we investigated the characterization of plasma DNA and it application to successfully detecting K-ras mutation at codon 12 in normal persons, hematopoietic neoplasms, and solid tumors. The range of plasma DNA in each group was 15.8 +/- 5.2 ng/ml, 43.3 +/- 29.8 ng/ml, and 26.8 +/- 17.0 ng/ml, respectively. The ranges in patients with solid tumor were gradually decreased to the normal level of around 15 ng/ml in 3 weeks postoperatively. Plasma DNAs consisted of about 200 bp DNA fragmentation and were convenient for PCR amplification of K-ras gene. The mutation at codon 12 by PCR-RFLP analysis was detected in 13(27%) of 49 patients with solid tumors such as pancreatic cancer, breast cancer, colon cancer, and gastric cancer. The diagnostic specificity was 100%. Serial observations by the PCR-RFLP analysis revealed disappearance of the mutant K-ras about 7 days after successful curative surgery in a patient with gastric cancer.     

胰腺胶样癌四例临床病理学分析

目的 探讨胰腺胶样癌的临床病理学特征、诊断、鉴别诊断及分子生物学特点.方法 分析4例胰腺胶样癌的临床特点,对标本进行病理形态学观察、免疫组织化学EnVision法和K-ras基因突变检测.结果 4例胶样癌中3例发生在胰头部,患者均为男性;另1例在胰体尾部,为女性;平均发病年龄为56.5岁.其中2例首发症状为腹痛,1例为尿糖增高,1例为查体发现.3例大体形态为囊实性结节,囊内含黏液,1例大体呈实性.低倍镜下,纤维及胰腺组织中可见边界清楚的黏液结节,大的黏液湖中可见纤细的纤维结缔组织间隔成多个小黏液湖;肿瘤细胞漂浮在黏液湖中,呈小巢或条索状,或腺管状,也可看到印戒细胞漂浮其中.3例癌周可见肠型胰腺导管内乳头状黏液性肿瘤(IPMN),仅例1伴发胰胆管型IPMN.免疫组织化学染色3例MUC2细胞膜阳性,1例MUC1阳性.3例中例1和例3发现K-ras基因突变,突变位点均位于12密码子Gly12Asp(GGT＞GAT)和Gly12Arg( GGT＞ CGT).结论 胰腺胶样癌是少见的胰腺导管腺癌亚型,经常伴发于IPMN和胰腺黏液性囊性肿瘤,应与普通胰腺导管腺癌、印戒细胞癌及假性囊肿等病变相鉴别.免疫组织化学MUC2多阳性表达,MUC1多为阴性,K-ras基因突变率较低.     

一株人胰腺癌细胞系的建立及其特性

人胰腺癌是很难建系的癌细胞之一,特别是从原发瘤建立的细胞系。我们成功地从一个胰腺癌组织建立了一株人胰腺癌细胞系,命名为PC-3。PC-3细胞呈上皮样,贴壁生长,经过四年连续培养,细胞系稳定。通过免疫组化、电镜观察、染色体及DNA含量分析,细胞集落形成及裸鼠移植,生长因子对瘤细胞生长的影响和癌基因的表达证实PC-3细胞系为人胰腺癌细胞系。PC-3细胞系的建立进一步丰富了人胰腺癌细胞库,对深入了解人胰腺癌细胞生物学及分子生物学特性提供了有力的物质基础。     

Diagnosis of pancreatic lesions using fine needle aspiration cytology: detection of K-ras point mutations using solid phase minisequencing.

AIMS--To improve the diagnostic value of fine needle aspiration biopsy of pancreatic lesions using a simple mutation detection method based on the polymerase chain reaction (PCR). METHODS--Fine needle aspirates from 21 suspected pancreatic lesions were analysed for K-ras codon 12 point mutations using solid phase minisequencing. RESULTS--A point mutation in codon 12 of the K-ras gene was detected in 14 of 17 cases of pancreatic carcinoma. No false positive results were recorded. The concordance of the result with routine cytology was 78%. All patients diagnosed as having malignant disease on cytology also had a K-ras point mutation. Additional information on the presence of malignancy was obtained using molecular genetic analysis in two cases. CONCLUSIONS--PCR based minisequencing is a promising method for the analysis of cytological material. K-ras point mutation analysis was modified to enable it to be carried out in a clinical laboratory. Advantages of the method include its simplicity and speed. Adequate sampling guidance is important but analysis can be performed even with small amounts of cellular material.     

ras gene mutations in salivary gland tumors

OBJECTIVE: To assess the prevalence of activating mutations in K-ras and H-ras genes in salivary gland tumors with ductal or acinar differentiation and to evaluate their potential correlation with clinical parameters. DESIGN: Paraffin-embedded tissue samples of salivary gland carcinomas were investigated by the application of a direct sequence analysis procedure with automated DNA sequencing of polymerase chain reaction-amplified ras sequences. SETTING: Tertiary care teaching hospital. PATIENTS: Twenty-four patients with salivary gland carcinoma were surgically treated. Nine had adenocarcinoma, 1 had adenosquamous carcinoma, 11 had mucoepidermoid carcinoma, and 3 had acinic cell carcinoma. RESULTS: Point mutations were detected in 7 (29%) of the 24 carcinomas examined. The K-ras gene was mutated in only 2 samples (8%): a GGC-to-ATC mutation at codon 13 in an adenocarcinoma and a GGC-to-GTC transversion mutation at codon 13 in a mucoepidermoid carcinoma. Five (21%) harbored H-ras mutations: 4 contained a GGC-to-GTC transversion mutation at codon 12 and 1 had 2 distinct mutations, the same G-to-T at codon 12 as was shown in the other cases and a GGT-to-GGA heterozygous mutation at codon 13. All the H-ras mutations were in the group of mucoepidermoid carcinoma lesions (45%; 5/11). CONCLUSION: Our data suggest that K-ras gene alteration is probably not an important factor in the oncogenesis of human salivary gland tumors. However, mutational activation of the H-ras gene appears to play a role in the development and/or progression of salivary gland mucoepidermoid carcinomas.     

Characterization of adenocarcinoma of the lung in a familial adenomatous polyposis patient

The incidence of several extracolonic tumors, such as duodenal carcinoma, is higher in familial adenomatous polyposis (FAP) patients than in the general population, but there is little information about lung carcinoma in FAP. A 43-year-old woman presented with a lung tumor 17 years after total colectomy for FAP. Pathohistological analysis of the lung tumor demonstrated mixed adenocarcinoma consisting of a papillary adenocarcinoma component and a bronchioloalveolar carcinoma component. Sequencing analysis indicated a germline APC mutation from TCA to TGA (stop) at codon 1110, but no pathogenic germline MYH mutations. The other APC allele in the lung carcinoma was not inactivated by somatic mutations, promoter methylation, or chromosomal deletion. No somatic mutations in any of the coding regions of the p53 gene or in the mutation hot spot regions of the K-ras or EGFR genes were detected in the carcinoma. Amplification, however, of three chromosome regions, 5p, 8q, and 12q14-12q21, was identified in the carcinoma on genome-wide high-resolution single-nucleotide polymorphism (SNP) microarray. The present results suggest that the chromosomal copy number alterations detected on SNP microarray were involved in the carcinogenesis of the adenocarcinoma of the lung in the present FAP patient.     

Analysis of K-ras oncogene mutation directly applied to atypical cell clusters on cytologic smear slides of bile and pancreatic juice

To develop an objective reference for the cytological evaluation of atypical cells in bile and pancreatic juice, we analyzed K-ras oncogene mutation In atypical cell clusters, which were collected directly from cytological smear slides; 50 samples (cell clusters) from 31 smear slides of 21 patients with carcinomas of the pancreatic head region, and nine samples from eight cases of benign disease. These cell clusters (5–1000 cells/cluster) were selectively suspended In buffer containing proteinase K, and subjected to DNA extraction. K-ras codon 12 mutation was determined by polymerase chain reaction amplification, followed by digestion with BstNI. The K-ras gene was amplified in 20 of 21 cases with carcinoma (34/50 samples), and In seven of eight cases with non-neoplastic disease (8/9 samples). Among the cases of which primary tumors showed K-ras mutation, amplification was successful in 10 of 11 cases; mutation was demonstrated in three of seven cases with cytologically atypical cells (4/11 samples), and in three of three cases with cytologically malignant cells (5/7 samples). No mutation was Identified in the 10 cases of carcinoma without K-ras mutation (0/15 samples), or in eight cases of non-neoplastlc disease (0/8 samples). Cytological details could be comparatively evaluated between atypical cell clusters with or without mutation on the same smear slides in two cases. This type of direct analysis of atypical cell clusters may be useful in the self-assessment of cytological diagnosis of bile and pancreatic juice.     

Medullary carcinoma of the pancreas in a man with hereditary nonpolyposis colorectal cancer due to a mutation of the MSH2 mismatch repair gene

Pancreatic adenocarcinoma has been reported in kindreds with hereditary nonpolyposis colorectal cancer (HNPCC). Medullary carcinoma of the pancreas is a recently described rare variant of pancreatic adenocarcinoma. We describe a man with colorectal carcinoma who subsequently developed pancreatic medullary carcinoma. The tumor displayed microsatellite instability and loss of expression of the mismatch repair proteins MSH2 and MSH6. Mutational analysis of the mismatch repair genes MLH1 and MSH2 demonstrated a pathogenic nonsense mutation within the MSH2 gene, which is consistent with a diagnosis of HNPCC. This report adds support to an association between HNPCC and pancreatic adenocarcinoma displaying the medullary phenotype, suggesting that medullary features in a pancreatic carcinoma may point toward a genetic cancer predisposition. To our knowledge, this is the first reported case of medullary carcinoma of the pancreas in a patient with HNPCC due to a mutation of the MSH2 gene.     

Adenocarcinoma arising in gastric heterotopic pancreas: clinicopathological and immunohistochemical study with genetic analysis of a case

Heterotopic pancreas in the stomach is a relatively common congenital condition, but the risk of malignant transformation is extremely low. In this study, we describe a case of adenocarcinoma arising from a gastric heterotopic pancreas and we consider its morphological and immunohistochemical features and genetic analysis, in order to examine its histogenesis. This unusual sequela was seen in a 57-year-old woman. Image studies showed a protruding lesion with a central ulcer located in the lesser curvature from the angle to the body of the stomach. A biopsy specimen confirmed this lesion as adenocarcinoma before total gastrectomy. The tumor showed mixed patterns of solid neoplastic-cell proliferation and moderately differentiated glandular structures, and also showed transitional lesions to obvious malignancy, that is, dysplasia, or adenocarcinoma in situ. Neoplastic cells had positive immunoreactivity for carbohydrate antigen (CA) 19-9, mucin (MUC) 1, and insulin, and the mutant allele-specific amplification method revealed a point mutation at K-ras codon 12 (GGT [Gly]-->GAT [Asp]), which is the most common mutational change observed in patients with pancreatic carcinoma. The features of the present case provide clear evidence that this tumor originated from heterotopic pancreatic tissue rather than from gastric epithelium.