多基因寡核苷酸芯片在胰腺癌及胰腺良性病变标本检测中的应用

目的 建立k-ras、p16、DPC4、p53基因寡核苷酸芯片对胰腺癌基因突变/缺失的检测系统并评估胰腺癌组织及胰腺良性疾病标本中多基因突变/缺失在临床检测中的应用.方法 采用双重或单独不对称PCR扩增16例胰腺癌组织及8例胰腺良性疾病标本中的目的 DNA.扩增产物加杂交液后与芯片进行杂交、清洗、扫描.结果 16例胰腺癌组织中12例可见k-ras突变,p16、DPC4、p53基因改变分别是7例.14例标本存在至少1个基因改变,其中5例标本存在1个基因改变,2例标本同时2个基因改变,4例标本存在同时3个基因改变,3例标本存在同时4个基因改变.8例胰腺良性疾病中只有3例显示k-ras、p16、p53、DPC4突变.结论 多基因芯片系统具有高度的灵敏性和准确性、快速简便、自动化程度高等优点,可同时检测胰腺癌k-ras、p16、DPC4和p53多个热点突变基因,可以高效地应用于胰腺癌基因突变的研究.     

石蜡包埋胰腺癌组织中DPC4第5/6、7、9、10外显子基因改变的检测

目的研究石蜡包埋胰腺癌组织中DPC4第5/6、7、9、10外显子基因的改变.方法用聚合酶链反应单链构象多态性分析(PCR-SSCP)银染技术检测46例石蜡包埋胰腺癌组织及癌旁正常胰腺组织中DPC4基因的缺失和突变.结果 5例胰腺癌未扩增出DPC4条带,DPC4基因第5/6、7、9、10外显子的纯合性缺失率为10.9%(5/46);1例胰腺癌可见异常泳动带,基因突变率为2.2%(1/46).结合以前实验,DPC4基因所有外显子的纯合性缺失率为39.1%(18/46),基因突变率为23.9%(11/46),总改变率为58.7%(27/46).27例有基因改变的胰腺癌除1例外均为中、低分化腺癌,中、低分化组与高分化组之间的差异有显著性(P<0.01).结论 DPC4作为一种抑癌基因,其改变在胰腺癌的形成中可能起重要的作用.DPC4基因改变与胰腺癌细胞分化程度密切相关.     

石蜡包埋胰腺癌组织中DPC4第5／6、7、9、10外显子基因改变的检测

目的研究石蜡包埋胰腺癌组织中DPC4第5／6、7、9、10外显子基因的改变。方法用聚合酶链反应单链构象多态性分析(PCR-SSCP)银染技术检测46例石蜡包埋胰腺癌组织及癌旁正常胰腺组织中DPC4基因的缺失和突变。结果 5例胰腺癌未扩增出DPC4条带,DPC4基因第75／6、7、9、10外显子的纯合性缺失率为10．9％(5／46)；1例胰腺癌可见异常泳动带,基因突变率为2．2％(1／46)。结合以前实验,DPC4基因所有外显子的纯合性缺失率为39．1％(18／46),基因突变率为23．9％(11／46),总改变率为58．7％(27／46)。27例有基因改变的胰腺癌除1例外均为中、低分化腺癌,中、低分化组与高分化组之间的差异有显著性(P<0．01)。结论 DPC4作为一种抑癌基因,其改变在胰腺癌的形成中可能起重要的作用。DPC4基因改变与胰腺癌细胞分化程度密切相关。     

k-ras基因寡核苷酸芯片的研制及在胰腺癌组织中检测应用研究

目的建立ras基因寡核苷酸芯片对胰腺癌基因突变的检测系统。评估16例胰腺癌组织k-ras基因12、13、61位密码子变突检测。方法采用双重或单独不对称PCR扩增标本中的目的DNA。扩增产物加杂交液后与芯片进行杂交、清洗、扫描。结果16例胰腺癌组织中k-ras为75%,突变都发生在12密码子,k-ras以12密码子第2位核苷酸突变发生率高(8/12)。结论k-ras基因芯片系统具有高度的灵敏性和准确性、快速简便、自动化程度高等优点,可同时检测胰腺癌k-ras多个突变位点基因,有利于临床应用。     

胰管刷检标本K-ras基因突变检测在胰腺癌诊断中的价值

目的 探讨胰管刷检标本K-ras基因突变检测在胰腺癌诊断中的价值。方法 应用突变富集聚合酶联反应（PCR）－单链构象多态性（SSCP）法，检测胰腺疾病胰管刷检标本K-ras基因第一外显子第12密码子点突变。结果 35例胰管刷检标本PCR扩增均获成功，成功率为100％。20例胰腺癌中14例K-ras突变（70％），7例慢性胰腺炎中1例K-ras突变（14％），两组间差异有显著性（P＜0．05）。胰腺囊腺瘤，十二指肠乳头癌均未见K-ras突变。胰管刷检标本K-ras突变与胰腺癌部位无关。胰管刷检K-ras突变检测诊断胰腺癌的敏感性，特异性和准确性分别为70％，90％和83％。结论 检测胰管刷检标本中K-ras基因突变有助于胰腺癌的诊断，具有良好的临床应用前景。     

非小细胞肺癌患者呼出气冷凝液和肿瘤组织中p16基因突变的研究

目的 研究呼出气冷凝液(EBC)和肺癌组织中p16基因突变,探讨EBC中检测的可行性和临床意义.方法 收集30例非小细胞肺癌(NSCLC)患者的肺癌组织和EBC标本,同期20名健康体检者的EBC标本作为对照.提取NSCLC患者手术切除的肺癌组织中的DNA,对β-actin基因片段扩增阳性的EBC标本和已提取的肺癌组织DNA进行p16基因1、2、3号外显子PCR扩增,并进行DNA基因测序,用DNASTAR软件进行突变比对,结果进行统计学分析.结果 ①30例NSCLC患者的EBC中β-actin基因片段扩增阳性26例,26例中有9例检出p16基因突变,突变率为34.6％;EBC中检出p16基因突变患者,其肺癌组织中均发现p16基因突变.②30例NSCLC癌组织中检测到p16基因突变15例,突变率为50.0％;癌旁组织均未检测到p16基因突变.③9例NSCLC患者EBC中p16基因突变的外显子为1号外显子3例,2号外显子5例,3号外显子1例;15例NSCLC患者肿瘤组织中p16基因突变的外显子为1号外显子4例,2号外显子8例,3号外显子3例.④26例NSCLC患者EBC中β-actin基因扩增阳性,Ⅰ期、Ⅱ期和Ⅲ期患者p16基因突变率分别为25.0％(3/12)、28.6％(2/7)和57.1％(4/7)( P＞0.05);鳞癌和腺癌患者的p16基因突变率分别为42.9％(6/14)和25.0％(3/12)(P＞0.05).⑤同一患者EBC与肺癌组织中p16基因突变的外显子种类、突变方式、突变类型和密码子均相同.结论 肺癌患者EBC和癌组织中均能检测到p16基因突变,并有高度的一致性.EBC中p16基因突变检测可作为一种简便、快速的肺癌诊断方法.     

PCR-MASA检测胰腺癌外周血K-ras基因点突变的研究

目的了解胰腺癌外周血中K-ras基因点突变检测的临床价值.方法采用PCR-MASA法检测胰腺癌患者外周血中K-ras基因点突变.结果胰腺癌外周血标本中K-ras基因点突变率为38.1%(8/21),而所有被检测的急、慢性胰腺炎、胰岛素瘤、壶腹癌、十二指肠乳头癌、胆管癌及胆石症患者外周血标本均无K-ras基因突变.结论(1)PCR-MASA方法简捷、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)对外周血标本检测K-ras基因第12位密码子有无突变,具有临床实用性,有助于判断胰腺病变良恶性及胰腺癌的早期诊断.     

DPC4基因与胰腺癌关系的研究进展

DPC4是一种肿瘤抑制基因 ,其编码的蛋白是TGF β信号传递的中心分子 ,与胰腺癌的发生关系密切。约有 5 0 %的胰腺癌存在DPC4缺失 ,且它的缺失多发生在III期和浸润性生长的胰腺癌。存在DPC4突变的胰腺癌患者生存期缩短。DPC4对胰腺癌的诊治可能有较为重要的临床意义。     

KAI1基因在胰腺癌中的突变分析

目的探讨KAI1基因在胰腺癌中的突变情况.方法对17例伴有淋巴结转移的原发性胰腺癌(Ⅲ期),7例无淋巴结转移的原发性胰腺癌(2例Ⅰ期,5例Ⅱ期)和9例正常胰腺组织标本进行KAI1基因突变分析.结果24例胰腺癌标本中,7例证实有KAI1点突变.这种突变出现在886位核苷酸上,导致从A到G的转变,缬氨酸置换异亮氨酸,且有突变的癌标本均属于晚期有转移的胰腺癌(Ⅲ期).结论上述结果提示KAI1基因突变使KAI1 mRNA水平降低可能是胰腺癌转移的主要因素之一.     

PCR—MASA检测胰腺癌外周血K—ras基因点突变的研究

目的 了解胰腺癌外周血中K—ras基因点突变检测的临床价值。方法 采用PCR—MASA法检测胰腺癌患外周血中K—ras基因点突变。结果胰腺癌外周血标本中K—ras基因点突变率为38．1％(8／21)，而所有被检测的急、慢性胰腺炎、胰岛素瘤、壶腹癌、十二指肠乳头癌、胆管癌及胆石症患外周血标本均无K—ras基因突变。结论 (1)PCR—MASA方法简捷、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)对外周血标本检测K—ras基因第12位密码子有无突变，具有临床实用性，有助于判断胰腺病变良恶性及胰腺癌的早期诊断。     

肿瘤抑制基因p16在胰腺腺癌细胞系中因纯合性缺失而失表达

目的：检测肿瘤抑制基因p16在胰腺腺癌细胞系中的基因改变和在mRNA以及蛋白质水平的表达。方法：使用PCR方法检测基因的缺失情况；使用单链构象多态性（SSCP）方法检测基因有无突变；使用甲基化特异性PCR（MSP）方法检测基因启动子区域的甲基化状态；使用RT－PCR方法检测基因在mRNA水平的表达；使用免疫细胞化学染色和Western blot方法检测基因在蛋白质水平的表达。结果：被检测的6个胰腺腺癌细胞系中，有3个细胞系存在p16基因的纯合性缺失，这3个细胞系在mRNA水平和蛋白质水平均无p16基因的表达。在全部6个细胞系中没有发现p16基因突变和启动子区域的过甲基化。结论：在被检测的6个胰腺腺癌细胞系中，纯合性缺失是造成p16基因失表达的原因。     

Loss of DPC4 expression and its correlation with clinicopathological parameters in pancreatic carcinoma

AIM: DPC4 is a tumor suppressor gene on chromosome 18q21.1 that has high mutant frequencies in pancreatic carcinogenesis. The purpose of this study was to investigate the role of DPC4 alterations in tumorigenesis and progression of pancreatic carcinomas.METHODS: We studied the immunohistochemical markers of DPC4 in 34 adenocarcinomas and 16 nonmalignant specimens from the pancreas. The 16 nonmalignant specimens from the pancreas included 8 non-neoplastic cysts and 8 normal pancreatic tissues. The relationship between DPC4 alterations and various clinicopathological parameters was evaluated by chi-square test or Fisher's exact test.Survivals were calculated using Kaplan-Meier method (by a log-rank test).RESULTS: All the 16 nonmalignant cases of the pancreas showed expression of DPC4 gene. Loss of DPC4 expression was seen in 8 of 34(23.5 %) pancreatic adenocarcinomas.The frequency of loss of DPC4 expression was higher in poorly differentiated adenocarcinoma (G3) than in well and moderately differentiated adenocarcinoma (G1 and G2)histologically (P=0.037). Loss of DPC4 expression of the patients at TNM stage Ⅳ was also higher than that of the patients at TNM stages Ⅰ, Ⅱ and Ⅲ (60.0 % at stage Ⅳ,versus14.3 % atstage Ⅰ, 18.2 % at stage Ⅱ, and 18.2 % at stage Ⅲ) (P=0.223). The mean and median survival in patients with DPC4 expression was longer than those in patients with loss of DPC4 expression. Kaplan-Meier survival analysis demonstrated patients with DPC4 expression had a higher survival rate than patients with loss of DPC4 expression, but the difference did not reach statistical significance (P =0.879).CONCLUSION: This study suggests that DPC4 is involved in the development of pancreatic carcinoma and is a late event in pancreatic carcinogenesis, DPC4 expression may be a molecular prognostic marker for pancreatic carcinoma.     

Loss of DPC4 expression and its correlation with clinicopathological parameters in pancreatic carcinoma

AIM: DPC4 is a tumor suppressor gene on chromosome 18q21.1 that has high mutant frequencies in pancreatic carcinogenesis. The purpose of this study was to investigate the role of DPC4 alterations in tumorigenesis and progression of pancreatic carcinomas.METHODS: We studied the immunohistochemical markers of DPC4 in 34 adenocarcinomas and 16 nonmalignant specimens from the pancreas. The 16 nonmalignant specimens from the pancreas included 8 non-neoplastic cysts and 8 normal pancreatic tissues. The relationship between DPC4 alterations and various clinicopathological parameters was evaluated by chi-square test or Fisher's exact test.Survivals were calculated using Kaplan-Meier method (by a log-rank test).RESULTS: All the 16 nonmalignant cases of the pancreas showed expression of DPC4 gene. Loss of DPC4 expression was seen in 8 of 34(23.5 %) pancreatic adenocarcinomas.The frequency of loss of DPC4 expression was higher in poorly differentiated adenocarcinoma (G3) than in well and moderately differentiated adenocarcinoma (G1 and G2)histologically (P=0.037). Loss of DPC4 expression of the patients at TNM stage Ⅳ was also higher than that of the patients at TNM stages Ⅰ, Ⅱ and Ⅲ (60.0 % at stage Ⅳ,versus14.3 % atstage Ⅰ, 18.2 % at stage Ⅱ, and 18.2 % at stage Ⅲ) (P=0.223). The mean and median survival in patients with DPC4 expression was longer than those in patients with loss of DPC4 expression. Kaplan-Meier survival analysis demonstrated patients with DPC4 expression had a higher survival rate than patients with loss of DPC4 expression, but the difference did not reach statistical significance (P =0.879).CONCLUSION: This study suggests that DPC4 is involved in the development of pancreatic carcinoma and is a late event in pancreatic carcinogenesis, DPC4 expression may be a molecular prognostic marker for pancreatic carcinoma.     

Methylation and mutation analysis of p16 gene in gastric cancer

AIM: To study methylation, frequencies of homozygous deletion and mutation of p16 gene in gastric carcinoma.METHODS: The methylation pattern in exon i and exon 2 of p16 gene was studied with polymerase chain reaction (PCR), using methylation sensitive restriction endonuclease HpaⅡ and methylation insensitive restriction endonuclease MspI. PCR technique was used to detect homozygousdeletions of exon 1 and exon 2 of p16 gene and single strand conformation polymorphism (SSCP) technique was used to detect the mutation of the gene.RESULTS: Hypermethylation changes in exon 1 and exon 2 of p16 gene were observed in 25 % and 45 % of 20 gastric cancer tissues, respectively, while no methylation abnormality was found in normal tissues. The homozygous deletion frequency of exon i and exon 2 of p16 gene in 20 gastric cancer tissues was 20 % and 10 %, respectively. No mutation was found in exon i of p16 gene, while abnormal single strands were found in 2 (10 %) cases in exon 2 as detected by SSCP.CONCLUSION: The results suggest that hypermethylation and abnormality of p16 gene may play a key role in the progress of gastric cancer. Hypermethylation of exon 2 of p16 gene may have effects on the carcinogenesis of gastric mucosa and may be a later event.     

Frequent deletions of tumor suppressor genes in pure pancreatic juice from patients with tumoral or nontumoral pancreatic diseases

Background/Aims: K-ras codon 12 mutation is the most frequent genetic alteration in pancreatic cancer. Sensitivity and specificity of K-ras are not high enough to detect all pancreatic cancers, especially at early stage. This study investigated whether detection of p16 and/or DPC4 deletions along with K-ras mutation in DNA samples could improve the definition of patients at risk of pancreatic cancer. Methods: K-ras mutations were investigated by sequencing. p16 and DPC4 homozygous deletions were studied using comparative multiplex polymerase chain reaction of DNA in pancreatic juice sampled during endoscopic retrograde pancreatography in 57 patients with either pancreatic cancer (group I, 18 patients), chronic pancreatitis (group II, 20 patients), or nontumoral pancreatobiliary disease (group III, 19 patients). Results: The frequencies of Ki-ras mutations were 61% in group I, 10% in group II, and 10.5% in group III. The frequencies of p16 exon 2 and DPC4 deletions were, respectively, 28 and 36% in group I, 50 and 58% in group II, and 24 and 36% in group III. Conclusions: The combination of p16 and DPC4 deletions with K-ras mutation does not improve the diagnosis of pancreatic cancer based on K-ras mutation alone. These data suggest that tumor suppressor gene inactivation can occur with a high frequency during nonmalignant pancreatic diseases.     

DPC4 gene mutation in colitis associated neoplasia.

BACKGROUND: Colitis associated dysplasia and cancer often have deletions involving the long arm of chromosome 18q, suggesting the location of a tumour suppressor gene critical for their tumorigenesis. The DPC4 gene, which is genetically inactivated in pancreatic and other cancers, has recently been described. AIM: Because DPC4 is located at 18q21.1, the hypothesis that it could be a mutated tumour suppressor gene in colitis associated neoplasms was tested. PATIENTS: Advanced neoplastic lesions from six patients having chronic colitis were analysed for DPC4. METHODS: Individual exons of DPC4 were amplified by the polymerase chain reaction (PCR) and sequenced from genomic DNA of tissue specimens dissected by cryostat. RESULTS: DPC4 was found to have biallelic inactivation in one of three neoplasms shown to have allelic loss of 18q. The mutation had been acquired somatically in a plaque of high grade dysplasia. The mutation created a non-sense codon, which would cause premature termination of protein translation. CONCLUSION: The DPC4 gene is a target of 18q LOH events in colitis associated neoplasia.     

初诊和复发的急性淋巴细胞白血病P15、P16基因失活的研究

目的：探讨多肿瘤抑制基因（MTS）P15和（或）P16的改变是否可影响急性淋巴细胞白血病（ALL）的病程及其预后。方法：跟踪30例复发的ALL患者在初诊和第1次骨髓复发时的骨髓标本，用PCR法监测P16 exon 1,P16 exon 2,P15 exon 1的缺失；用REP法检测P16 exon 1,P15 exon 1高度甲基化情况。结果：在初诊患者中共有50％（12／30）的患者有（或）P16基因失活，P16缺失的患者有8例，其中7例伴有P15缺失，7例P15甲基化的患者有2例伴P16甲基化。在初诊时，15例无该基因异常的患者，复发期间有11例出现P15和（或）P16异常，占86．7％（26／30），与初诊时48％（12／25）比较，差异有极显著性意义（P＜0．01）。结论：我们认为P15和（或）P16的失活可加快疾病的复发，监测ALL患者的P15和（或）P16失活，可用于预测疾病的病程和预后，初诊和复诊均有异常者为难治性疾病。