大肠癌p53、K-ras基因突变研究

目的：探讨p53,K-ras基因突变与大肠癌发生、发展的关系。方法：应用PCR－SSCP方法研究68例大肠癌和癌旁组织以及20例正常组织p53,K-ras基因突变情况。结果：大肠癌组中p53，K-ras基因突变率分别为47．1％和44．1％，明显高于癌旁组（分别为13．2％和7．4％），20例正常组织中未检出p53、K-ras基因突变，大肠癌伴有淋巴结转移及远处转移，p53、K-ras基因突变率明显高于无淋巴结及远处转移；p53、K-ras基因突变与组织学分型无关。结论：p53、K-ras基因突变与大肠癌发生、发展有密切关系，在细胞癌变中起重要作用，可作为评估大肠癌转移的分子生物学指标。     

p53基因突变及p53,APC基因缺失与胃癌关系的研究

为明确ｐ５３基因突变，缺失，ＡＰＣ基因缺失在胃癌发病机制中的作用，应用ＰＣＲ－ＳＳＣＰ方法对抑癌基因ｐ５３第４，５，６，７，８外显子，第６内含子在８７例胃癌及癌前病变中的突变规律以及ＰＣＲ－ＲＦＬＰ方法对ｐ５３基因第４外显子，第６内含子，ＡＰＣ基因在２５对胃癌及癌旁组织的杂合缺失规律进行了探讨，结果发同，ｐ５３突变率在肠化，不典型增生，胃癌分别为３７．５％（３／８），４２．１％（８／１９），５３．     

PCR—SSCP技术快速检测结核分枝杆菌rpoB基因突变的研究

目的 应用ＰＣＲ－ＳＳＣＰ技术直接快速检测痰标本中结核分枝杆菌ｒｐｏＢ基因突变,评价此方法用于结核分枝杆菌利福平（ＲＦＰ）耐药性的意义。方法 以结核分枝杆菌Ｈ３７Ｒｖ为对照、５０例耐ＲＦＰ（单耐和多耐）结核分枝杆菌临床分离株、１２例ＲＦＰ敏感菌株、３５例耐ＲＦＰ肺结核患者痰标本,采用ＰＣＲ－ＳＳＣＰ检测痰标本和菌株中结核杆菌ｒｐｏＢ基因突变。结果 ５０例耐ＲＦＰ菌株中有４５例ＰＣＲ－ＳＳＣＰ条带与     

PCR—SSCP方法用于痰标本中结核分支杆菌rpoB基因突变检测

本文用ＰＣＲ－ＳＳＣＰ方法直接检测痰标本中结核分支杆菌ｒｐｏ基在变，评价此方法用于结核分支杆菌利福平耐性试验的意义，以期建立一种直接检测分支杆菌耐利福平的快速方法。本文用ＰＣＲ－ＳＳＣＰ方法分析了３０例结核病人和２０例非结核性肺部疾病病人痰标本。３０份肺结核病人的痰用ＰＣＲ－ＳＳＣＰ检测痰中结核分支杆菌ｒｐｏＢ基因突变：２１份痰ＰＣＲ扩增阳性，其中１３份ＳＳＣＰ图谱与参考株Ｈ３７ＲｖＳＳＣＰ图谱有     

一种简单迅速筛选肿瘤基因组中基因突变的方法

目的 建立一种新的非同位素ＳＳＣＰ分析方法。方法 常规提取人类基因组ＤＮＡ作为模板,ＰＣＲ扩增ｎｍ２３－Ｈ１基因,变性ＰＣＲ产物,６％非变性聚丙烯酰胺凝胶ＳＳＣＰ电泳,银染后分析结果。结果 改进了目前ＰＣＲ－ＳＳＣＰ分析中应用同位素标记的检测方法,采用银染方法,建立起简单迅速的非同位素ＰＣＲ－ＳＳＣＰ分析方法和适宜条件。并将这种方法成功地应用于肿瘤转移抑制基因点突变的检测。结论 非同位素聚合酶链反     

PCR—SSCP技术快速检测结核分枝杆菌三种基因突变

聚合酶链反应-单链构象多态性(PCR-SSCP)是快速检测基因突变的重要方式之一[1].应用此法对辽宁省90例涂阳患者痰标本分离结核分枝杆菌进行rpoB、rpsL、56例katG基因突变检测,与传统耐药结果比较分析,寻找结核分枝杆菌耐药性与基因突变的关系.     

大肠癌中p53基因突变与HPV研究进展

ｐ５３基因和人乳头瘤病毒（ＨＰＶ）是目前肿瘤病因学领域研究的热点。本文对大肠癌中ｐ５３基因突变和ＨＰＶ的感染情况进行了综述，探讨了这两种因素的单独作用及协同作用在大肠癌癌变过程中可能所起的作用，它的阐明为进一步理解和探寻大肠癌的发病机制提供了新的线索，对大肠癌的防治将起到一定的指导作用。     

p53 gene mutations and expression of p53 and mdm2 proteins in invasive breast carcinoma

The aim of the study was to analyzep53 gene mutations and the expression of p53 and mdm2 proteins in 31 randomly selected invasive breast carcinomas. The results were then correlated with tumor grade, stage, estrogen receptor status, nodal status, and DNA ploidy. The expression of the proteins p53 and mdm2 was determined immunohistochemically using formalin-fixed, paraffin-embedded material. Screening for p53 mutation involved analysis of the highly conserved regions of thep53 gene (exons 5–9) by the polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) technique. PCR products with band shifts were directly sequenced. Immunohistochemical staining of p53 was positive in 9 cases (29.0%), only 2 of which showed ap53 gene mutation. These were identified as a CG transversion at the second position of codon 278 in exon 8 and an AG transition at the second position of codon 205 in exon 6. A third case with a mutation was observed (CT transition, position 1 of codon 250 in exon 7) that did not show p53 immunohistochemically. Of the 9 p53-positive tumors, 2 were moderately differentiated (grade II). The remaining tumors were poorly differentiated (7/9). By contrast, p53-negative carcinomas were well differentiated (grade I) in most cases (P=0.02). DNA cytometry in 8 of the 9 p53-positive carcinomas revealed an aneuploid stem line. The majority of the p53-negative tumors were diploid (P=0.01). Mdm2 oncoprotein was detected in 10 tumors (32.2%), 4 of which were p53-positive, including the 3 with mutations. The grading of the mdm2-positive tumors was moderate or poor, G1 carcinomas were always noted to be mdm2-negative (P=0.04). Overexpression of p53 protein is a complex mechanism and does not merely indicate the detection of mutations in thep53 gene. This study has shown that p53 expression correlates with tumor grade and DNA ploidy. Mdm2 expression was also associated with the tumor grade. Immunohistological demonstration of the p53 protein alone is insufficient as a basis for comment on the functional state of thep53 gene and gene product. The interrelation between recognition of the p53 protein and gene mutation needs more careful assessment to define their roles in the control of neoplasia.     

Detection of p53 mutations using nonradioactive SSCP analysis: p53 is not frequently mutated in myelodysplastic syndromes (MDS)

Summary p53 is one of the most frequently mutated genes in human cancers. Since p53 has been implicated in lymphatic and some myeloid leukemias, such as the blastic phase of chronic myelogeneous leukemia, we sought to address the role of p53 gene mutations within exons 4–9 in myelodysplastic syndromes (MDS), a myeloid preleukemic condition. In order to avoid the potential hazard of using radioactive single-strand conformation analysis (SSCP), we used a nonradioactive SSCP method based on the silver stain of small minigels. In cell lines with known point mutations of the p53 gene, aberrant migrating bands were found. Serial dilutions indicated a sensitivity comparable to radioactive methods. Furthermore, a common polymorphism within the 4th exon of the p53 gene was easily detected. However, of 17 primary samples from patients with MDS, none harbored a p53 gene mutation. We conclude that this nonradioactive method can easily be used to screen for p53-gene mutations, and that p53-gene mutations do not play a major role in the pathogenesis of MDS.     

肝细胞癌基因P53异常的研究

目的：分析我国重庆地区肝细胞癌Ｐ５３基因失活机制及突变谱。方法：采用ＰＣＲ—ＲＦＬＰＰＣＲＳＳＣＰ和ＰＣＲ直接测序技术对来自我国重庆地区２８例肝细胞癌Ｐ５３抑癌基因结构异常进行了分析。结果：６１．５１％的肝癌存在ｐ５３的杂合缺失：５０％肝癌伴有ｐ５３基因突变，其突变模式为突普通散在于５６７和８外显子，其中第７外显子２４９们密友情子突弯率最高（２１％）；具有突变的肝癌多同时伴夺缺失。伴有ｐ５３基因结构异常的肝癌均属进展期。结论：我国重庆地区肝癌存在Ｐ５３基因结构异常，Ｐ５３基因结构异常，ｐ５３基因突变模式反映了该地区肝癌发生可能与肝炎病互和黄贡互素两种因素及其相互作用有关；ｐ５３基因结构异常属肝癌晚期事件，可能参加与肝癌的进展过程。     

原发性胃癌p53基因突变

目的 p53基因是当前抑癌基因研究中的热点之一。迄今,有关 p53基因异常与胃癌临床病理学参数如大体类型、临床分期、组织分化程度,浸润深度及淋巴结转移之间的关系尚无定论。Tumura 报告p53基因改变主要发生于异倍体瘤,国内尚无报道。本实验目的主要是分析中国人原发性胃癌 p53基因突变与这些病理参数,包括 DNA 倍体之间的关系。方法用聚合酶链式反应—单构象多态分析(PCR—SSCP)技术对20例原发性胃癌 p53基因外显子5—8突变进行检测。结果 8例(40%)发生了突变,其中2例发生在外显子7,4例发生在外显子8。0至Ⅲ期均有突变存在。66.7%(6/9)的异倍体瘤检测到了p53突变,而二倍体瘤中只有18.2%(2/11)发生了 p53突变。结论 p53基因突变与胃癌临床病理参数如大体类型、分期、组织分化程度、浸润深度及淋巴结转移之间无明显关系,而与胃癌 DNA 倍体改变有关。     

p53 gene mutations in soft-tissue sarcomas-correlations with p53 immunohistochemistry and DNA ploidy

The significance ofp53 mutations in a group of 67 soft-tissue tumors was examined using single-strand conformation polymorphism and direct sequencing analysis. Molecular findings were correlated with immunohistochemical detection of thep53 protein and DNA ploidy status. Mutations of thep53 gene were detected in 13 (19.5%) out of 67 cases of soft-tissue tumors. Only there were localized outside the conservative regions of thep53 gene. Six mutations were described for the first time in these tumors. Most of the mutations were point mutations in exons 5–8 and, in one case, a deletion at the 3-splice site of exon 5 could be demonstrated. There was no significant correlation between the occurrence ofp53 mutations and the histological grade, although a high number of mutations were defined in poorly differentiated tumors (grade 3). Molecular finding of ap53 gene mutation and immunohistochemical detection ofp53 expression did not correlate, which may be due to the high percentage of nonsense mutations in our study (50%). We confirm that only DNA sequencing allows a unique identification and differentiation of mutations in thep53 gene. Other factors may be responsible for the detection ofp53 protein in many cases. Histological grade correlated with aneuploidy. The frequency of mutations observed was in accordance with values quoted in the literature. Generally,p53 mutations andp53 overexpression are more likely to represent a late event in the oncogenesis of soft-tissue tumors.     

p53 overexpression and K-ras gene mutations in primary sclerosing cholangitis-associated biliary tract cancer

Cholangiocarcinoma occurs frequently in patients with primary sclerosing cholangitis (PSC). We evaluated the incidence and prognostic significance of p53 protein overexpression and K-ras gene mutations in patients with biliary tract cancer and PSC. p53 protein expression was determined in specimens from 12 patients with biliary tract cancer, using the antibody, D07. K-ras mutations were detected using DNA sequencing and a mutation ligation assay. Accumulation of p53 protein was detected in 6 of 12 tumors (50%). K-ras mutations were detected in 4 of 12 tumors (33%). Overall survival in patients with p53-negative tumors was significantly longer (P < 0.05) than that in patients with p53-positive (mutant) tumors. Similarly, overall survival was significantly longer (P < 0.05) in the absence of a K-ras mutation than in patients with a tumor containing a K-ras mutation. Mean interval from the time of diagnosis of PSC until the diagnosis of biliary tract cancer was significantly shorter (P < 0.05) in patients with p53 overexpression than in those patients without p53 overexpression (2 versus 47 months). p53 overexpression and K-ras mutations occur commonly in patients with PSC and biliary tract cancer and are associated with a shortened survival. Patients with longstanding PSC are less likely to have these genetic alterations and may have a better prognosis.     

N-ras and p53 gene mutations in Japanese patients with myeloproliferative disorders

Alterations of the N-ras oncogene and p53 tumor suppressor gene have been demonstrated to play an important role in pathogenesis of hematological malignancies. We simultaneously investigated genetic lesions of both genes in bone marrow cells from 64 Japanese patients with myeloproliferative disorders (MPD), including polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (MF), by direct sequencing analysis. No mutations of the N-ras gene were detected in any cases. Two patients, one with chronic neutrophilic leukemia derived from PV and one with acute mylogenous leukemia derived from ET, exhibited three mutations of the p53 gene. Among them, two were missense mutations in exon 5 or 7 and one was a deletion in exon 5. All samples in chronic phase or from MF were devoid of mutations in both genes. These data suggested that disruptions of both genes are extremely rare in MPD in chronic phase and that loss of functions in the p53 gene could be involved in progression of MPD such as PV and ET.     

介导野生型p53基因转移的重组腺病毒的构建研究

应用同源重组方法成功地构建了携带野生型ｐ５３基因的复制缺陷型重组腺病毒，通过光镜、电镜、酶切、ＰＣＲ共扩增等方法证实了构建载体的正确性，并使常规的同源重组方法进一步改进，简化了载体构建的操作程序，结果准确，成功率高。本结果可用于以腺病毒为载体进行肿瘤、心血管疾病等基因治疗的研究。     

毛细管电泳和聚丙烯酰胺电泳法检测肺癌组织p53基因突变效果分析

目的筛选检测肺癌组织p53基因突变的优良方法,为临床早期诊断肺癌提供依据。方法分别运用毛细管电泳（CE）与聚丙烯酰胺电泳（PAGE）-限制性片段长度多态性（RFLP）两种方法对38例肺癌及癌旁正常组织p53基因第249位密码子点突变进行检测。结果RFLP—CE可检测到p53基因第249位密码子的野生型26bp片段,用时〈20rain;RFLP—CE和RFLP-PAGE均能检测到基因突变,对29例非小细胞肺癌组织p53基因第249位密码子检测的突变率分别为31．03％（9／29）、20．69％（6／29）,P〈0．05。结论RFLP-EE可为大规模进行肺癌的早期诊断提供简便可靠的方法。