胃癌组织ras族基因突变与预后的关系

应用多聚酶链延伸反应－限制性片段长度多态笥分析法（PCR－RFLP）对88例福尔马林液固定，石蜡包埋胃癌组织C－Ha-ras第12位和13位，K－ras第12位和13位及N－ras第12位密码子的点突变进行检测，结果发现ras基因总突为18．2％（16．88），以c-Ha-ras第12位密码子突变率最高（13．6％），点突变的发生与肿瘤浆膜浸润，淋巴结转移，临床病理分期及术后生存期密切相关。以上结果提示，检测胃癌组织ras族基因点突变有助于判断胃癌患者的预后。     

胃癌组织ras基因群点突变及其一患者预后的关系(英文)

目的研究ｒａｓ基因点突变与胃癌患者预后的关系．方法应用多聚酶链延伸反应—限制性片段长度多态性分析法（ＰＣＲ－ＲＦＬＰ）对８８例福尔马林固定，石蜡包埋胃癌组织ＣＨａｒａｓ第１２位、６１位，Ｋｒａｓ第１２位、１３位和Ｎｒａｓ第１２位密码子的点突变进行检测．结果ｒａｓ基因群总突率为１８２％（１６／８８），点突变的与肿瘤浆膜浸润、淋巴结转移、临床病理分期及术后的生存期密切相关．结论检测胃癌组织ｒａｓ基因群点突变有助于判断胃癌患者的预后．     

胃幽门螺杆菌与胃癌发病机制关系的分子生物学研究

近期流行病学研究已发现胃幽门螺杆菌(HP)是胃癌发生的重要始发因素。本文应用敏感特异的聚合酶链反应(PCR)方法检测胃粘膜组织HP,分析其与C—Ha—ras第12位密码子点突变,ras基因产物P~(21)蛋白表达及DNA倍体的关系。发现C—Ha—ras点突变率于HP阳性组高于阴性组,并且HP阳性组较HP阴性组出现rasP~(21)表达增强,说明HP感染与C—Ha—ras基因活化有关,HP感染后DNA含量及S％期细胞明显增高,提示核苷酸代谢旺盛,DNA损伤及非整倍体的危险性也增高。本研究为HP致胃癌的可能机制从分子水平及细胞代谢水平作了初步研究。     

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。     

大肠癌患者血浆DNA中K-ras癌基因突变的检测

目的 评价血浆DNA中K－ras癌基因第12密码子点突变作为肿瘤标记物的临床应用价值。方法 用引物序列特异性聚合酶扩增链式反应（PASP）检测了32例大肠癌患者肿瘤组织DNA、血浆DNA中K－ras癌基因第12密码子点突变，对所有PASP法扩增得到的含点突变的PCR产物进行Sanger双脱氧链终止法测序。结果 14例（44％）大肠癌肿瘤组织DNA中存在K－ras癌基因第12密码子点突变；其中的13例（93％）在血浆DNA中存在与其肿瘤组织DNA相同类型的基因点突变。对于18例肿瘤标本DNA未见突变的各期大肠癌及5例正常献血员对照组，在其血浆DNA中也无一发现K－ras基因突变。结论 血浆DNA中K-ras癌基因第12密码子点突变有望成为一个肿瘤标志物应用于大肠癌的诊断。     

胰腺癌ras p21蛋白表达与AgNORs计数及组织学分级的关系

目的研究胰腺癌ras p21蛋白表达与AgNORs计数及组织学分级的关系。方法用免疫组化法检测了50例胰腺癌组织及50例正常胰腺组织中ras p21蛋白的表达；AgNORs染色技术检测胰腺癌细胞的AgNORs含量；观察肿瘤的组织学分级。结果ras p21蛋自在胰腺癌组织和正常胰腺组织中的表达有显著差异；ras p21蛋白阳性胰腺癌的AgNORs计数比阴性的AgNORs计数明显增多（P〈0．001）：ras p21蛋白表达与肿瘤组织学分级呈显著负相关（r5==0．99，P〈0．001）。结论胰腺癌的发生发展过程包含了ras p21基因的突变。ras p21蛋白表达可反映胰腺癌的恶性程度，是一个有效的判断胰腺癌预后的指标。     

ras和nm23的表达与胃癌转移的相关性研究

目的:观察nm23与ras在原发性胃癌中的表达水平与胃癌淋巴结转移的关系.方法:免疫组化和RT-PCR.结果:通过对80例原发性胃癌的研究表明nm23低表达者淋巴结转移率(90%)比nm23正常表达者高(40%,P<0.05).同时发现p21 ras阳性为65%,p21 ras阳性者淋巴结转移率高于p21 ras阴性者淋巴结转移率(78.5%对57.1%,p<0.05).肿瘤组织如同时具有nm23低表达和ras过表达则有更高的淋巴结转移率(94.12%),而nm23高表达和ras低表达者则淋巴结转移率更低(25%),ras-mRNA半定量分析说明ras的表达与淋巴结转移正相关,结论:nm23低表达和ras过表达在原发性胃癌淋巴结转移中起重要的联合作用.它们可以作为判断胃癌预后的一个生物学标志.     

支气管肺泡灌洗液中K-ras基因突变的检测在周围型肺癌诊断中的应用

目的　探讨支气管肺泡灌洗液 (BALF)中K ras原癌基因点突变在周围型肺癌诊断中的价值。方法　应用聚合酶链反应结合限制性片段长度多态性分析法 (PCR RFLP) ,检测 5 4例肺癌及 2 0例肺良性疾病患者支气管肺泡灌洗液中K ras基因第 12位密码子突变的情况。结果　肺癌及肺良性疾病中K ras基因突变率分别是3 5 2 % ( 19 5 4)和 5 0 % ( 1 2 0 ) ,非小细胞肺癌 (NSCLC)突变率明显高于小细胞肺癌 (SCLC) ,其中以肺腺癌突变率最高 ( 47 6% )。突变的发生与肿瘤TNM分型无关。结论　BALF中K ras基因突变的检测对肺癌有一定的早期诊断价值 ,可以协助纤维支气管镜提高对周围型肺癌的诊断率 ,尤其是对肺腺癌     

RASAL1表达及ras活性与胃肠道肿瘤临床关系的研究进展

大约有20％的人类肿瘤与ras的突变有关。ras蛋白调控细胞的一系列生物学行为,包括细胞增殖、分化、生存及凋亡等。ras蛋白属于小GTP结合蛋白,通过响应细胞外信号产生活性。RASAL1是一种rasGTP酶激活蛋白,可以抑制ras的活性。调节ras以及RASAL1的活性,可能是肿瘤靶向治疗的方向之一。检测ras以及RASAL1的活性有助于胃肠道肿瘤的早期诊断以及预后的判断。本文从ras的结构、功能及突变与肿瘤的关系,RASAL1作为ras的效应分子抑制ras的活性,ras、RASAL1与胃肠道肿瘤的关系三方面来综述了RASAL1表达及ras活性与胃肠道肿瘤临床关系的研究进展。     

线粒体突变及不稳定在胃癌中的研究

目的 探讨线粒体突变及不稳定在胃癌发生、发展中的作用.方法 利用激光显微切割技术分离胃癌组织及其切缘的正常组织,应用变性高效液相色谱DHPLC对胃癌线粒体D-loop调控区D-loop-(CA)n进行突变筛查及测序分析,同时进行线粒体D-loop非编码区(CA)n重复序列的不稳定(mtMSI)检测.结果 胃癌组织样本中D-loop-(CA)n调控区基因突变率为50.8％ (31/61),正常组织均未见有序列改变.29.5％ (18/61)发生线粒体重复序列(CA)n的不稳定.18例线粒体不稳定(mtMSI)中有16.4％(10/61)同时发生D-loop点突变,有8例同时存在核不稳定(nMSI-H).结论 胃癌mtDNA异常参与了肿瘤的发生、发展.     

应用多种方法检测胃癌演变过程中ras基因的突变

为了解胃癌演变过程中相关基因ｒａｓ的变化，应用聚合酶链反应（ＲＣＲ）－限制性片段长度多肽性分析法（ＲＦＬＰ），单链构象多肽性分析法，ＰＣＲ－ＤＮＡ测序的方法对中国人胃癌及癌前病变中的ｒａｓ基因家族变化规律进行了探讨。结果发现：Ｈ－ｒａｓ１２位点的突变率在肠化、不典型增生、进展期胃癌分别为１６．７％（６／３６），３１．２％（１５／４８），３４．７％（２５／７２）。在正常对照组、浅表性胃炎组均未发现Ｈ     

HMLH1 gene mutation in gastric cancer patients and their kindred

AIM: To study the status of hMLH1 gene point mutations of gastric cancer kindreds and gastric cancer patients from northern China, and to find out gene mutation status in the population susceptible to gastric cancer. METHODS: Blood samples of 120 members from five gastric cancer families, 56 sporadic gastric cancer patients and control individuals were collected. After DNA extraction, the mutations of exon 8 and exon 12 of hMLH1 gene were investigated by PCR-SSCP-CE, followed by DNA sequencing. RESULTS: In the five kindreds, the mutation frequency was 25% (5/16) for the probands and 18% (19/104) for the non-cancerous members, which were significantly higher than the controls (P<0.01 X2= 7.71, P<0.01 X2= 8.65, respectively). In the sporadic gastric cancer, the mutation frequency was 7% (4/56), which was similar to that (5/100) in the healthy controls. The mutation point of exon 8 was at 219 codon of hMLH1 gene (A-G), resulting in a substitution of Ile-Val (ATC-GTC), whereas the mutation of exon 12 was at 384 codon of hMLH1 gene (T-A) resulting in a substitution of Asp-Val (GTT-GAT), which were the same as previously found in hereditary nonpolyposis colorectal carcinoma. CONCLUSION: The members of gastric cancer families from northern China may have similar genetic background of hMLH1 gene mutation as those of hereditary nonpolyposis colorectal carcinoma.     

PCR-SSCP-DNA sequencing method in detecting PTEN gene mutation and its significance in human gastric cancer

AIM： To discuss the possible effect of PTEN gene mutations on occurrence and development of gastric cancer. METHODS： Fifty-three gastric cancer specimens were selected to probe PTEN gene mutations in genome of gastric cancer and paracancerous tissues using PCR-SSCP-DNA sequencing method based on microdissection and to observe the protein expression by immunohistochemistry technique. RESULTS： PCR-SSCP-DNA sequencing indicated that 4 kinds of mutation sites were found in 5 of 53 gastric cancer specimens. One kind of mutation was found in exons. AA-TCC mutation was located at 40bp upstream of 3＇ lateral exon 7 （115946 AA-TCC）. Such mutations led to terminator formation in the 297th codon of the PTEN gene. The other 3 kinds of mutation were found in introns, including a G-C point mutation at 91 bp upstream of 5＇ lateral exon 5（90896 G-C）, a T-G point mutation at 24 bp upstream of 5＇ lateral exon 5 （90963 T-G）, and a single base A mutation at 7 bp upstream of 5＇ lateral exon 5 （90980 Adel）. The PTEN protein expression in gastric cancer and paracancerous tissues detected using immunohistochemistry technique indicated that the total positive rate of PTEN protein expression was 66% in gastric cancer tissue, which was significantly lower than that （100%） in paracancerous tissues （P 〈 0.005）. CONCLUSION： PTEN gene mutation and expression may play an important role in the occurrence and development of gastric cancer.     

H-ras protooncogene mutations in human thyroid neoplasms

Structural alterations of protooncogene sequences may be involved in the pathogenesis of human neoplasms. We screened 54 thyroid tumors (36 benign and 18 malignant) for gene rearrangements of the protooncogenes c-myc, c-myb, c-fos, c-erb-B1, c-erb-B2, c-erb-A, N-ras, K-ras, and H-ras. Only mutations of H-ras were observed. None of the 15 colloid adenomas examined had detectable H-ras rearrangements. Of the remaining tumors, we observed mutations of H-ras in 4 benign and 4 malignant neoplasms. Gene amplification was found in 5 tumors. An aggressive recurrent papillary carcinoma had a marked amplification of one of the H-ras alleles. The amplified allele was truncated, in that the 3' variable tandem repeat was not a part of the amplification unit, and contained a codon 12 point mutation leading to a valine for glycine substitution. We also observed the association of low copy gene amplification with a codon 12 valine for glycine mutation in a follicular adenoma. Two tumors contained H-ras EcoRI polymorphisms not present in the DNA of normal thyroid from the same individuals, and one follicular carcinoma showed loss of an H-ras allele. Ras protooncogenes may become transforming by quantitative mutations, leading to increased expression, or qualitative mechanisms, through activating point mutations. Both of these appear to coexist in thyroid neoplasms, and it may be that a combination of both mechanisms is capable of inducing a more complete spectrum of neoplastic phenotypes.     

Relationship between inactivation of p16 gene and gastric carcinoma

AIM: To investigate the relationship between inactivation of p16 gene and gastric carcinoma, and the mechanism of inactivation of p16 gene in gastric carcinogenesis. METHODS: 40 fresh tumor tissue specimens were taken from primary gastric cancer patients. Expression of P16 protein was detected by immunohistochemical method. Deletion and point mutation of p16 gene were analyzed by polymerase chain reaction (PCR) and DNA sequencing, respectively. RESULTS: The frequency of loss of P16 protein expression in the gastric cancer tissue, adjacent nontumor tissue, and distal normal tissue was 77.5 % (31/40), 55.0 % (22/40), and 17.5 % (7/40), respectively (P<0.005). Homozygous deletion of exon 1 and exon 3 was observed in two and three cases, respectively, giving an overall frequency of homozygous deletion of 12.5 %. All five cases had diffuse type gastric carcinoma. No p16 gene point mutation was detected. CONCLUSION: These findings suggest a close correlation between inactivation of p16 gene and gastric carcinoma. Further investigations are needed to testify the mechanism of inactivation of p16 gene in gastric carcinogenesis.     

Impact of loss of heterozygosity of encoding phosphate and tensin homolog on the prognosis of gastric cancer

BACKGROUND AND AIM: Encoding phosphate and tensin homolog (PTEN) is a cancer suppressor gene and it has been assumed that gene mutation and loss of heterozygosity (LOH) occurs frequently in various types of carcinoma. However, the role of LOH of PTEN and its outcome variables in gastric cancer have not been well established. In the present study, we investigated the roles of PTEN, LOH and their outcomes. METHODS: Fresh frozen tumor samples from 119 gastric cancer patients with a primary diagnosis of gastric carcinoma were evaluated for LOH of PTEN using an automated sequencer. Results were compared with pathological parameters. The median follow-up period was 559 days. RESULTS: Loss of heterozygosity of PTEN was observed in 17.1% of patients (13/76) diagnosed with gastric cancer. No particular relationship was found with any clinicopathological factor. However, the prognosis of patients with LOH of PTEN was significantly poor. Multivariate analyses revealed that vascular invasion, invasion depth, LOH of PTEN, histology and lymph node metastasis were correlated with survival of the patient. CONCLUSIONS: Even though mutation of PTEN in gastric cancer has rarely been reported, according to our findings, LOH of PTEN frequently occurs in gastric cancers and is correlated with disease-related deaths. The LOH of PTEN is an independent prognostic factor and PTEN is a candidate as a haploinsufficient tumor suppressor in gastric cancers.     

Helicobacter pylori infection affects the expression of PCNA, p53, c-erbB-2 and Bcl-2 in the human gastric mucosa.

OBJECTIVE: Helicobacter pylori infection has been related to gastric carcinogenesis. This association is based on epidemiological data, pathological changes observed in the gastric mucosa, and chemical products from bacteria that may induce damage of DNA. In the present study we examined gastric endoscopic biopsies from patients with chronic gastritis, with and without H. pylori infection, and surgical biopsies from gastric cancer patients to evaluate whether this bacteria may induce changes in the expression of molecular markers associated with carcinogenesis. PATIENTS AND METHODS: the study involved 57 biopsies from the antral region of the stomach of patients with chronic gastritis and gastric cancer that were analyzed by immunohistochemistry. Molecular markers examined were: PCNA (Proliferating Cell Nuclear Antigen), p53, c-erbB-2, Bcl-2, and p21 H-ras. RESULTS: PCNA content of epithelial cells was significantly higher in H. pylori infected biopsies. Treatment aimed to eradicate H. pylori decreased the level of PCNA-positive cells in the group of patients that became H. pylori-negative as well as in H. pylori-positive patients. Nuclear p53 expression (used here as a surrogate marker for p53 mutation/inactivation) and c-erbB-2 expression were observed only in the group of patients that remained with the bacteria after treatment. A higher bcl-2 expression in lymphoid cells was observed in H. pylori-positive biopsies, and treatment did not change the expression of this protein. No significant expression of p21 H-ras was observed in the studied biopsies. CONCLUSION: this study suggests that H. pylori is involved in the induction of molecular changes that might predispose human gastric mucosa cells to pre-neoplastic and neoplastic events.     

Clarithromycin-resistance and point mutations in the 23S rRNA gene in Helicobacter pylori isolates from Japan

BACKGROUND: Resistance of Helicobacter pylori to clarithromycin is mostly due to the point mutations in the 23S rRNA. In Japan, however, the frequency of these mutations has not been fully investigated. Furthermore, no study has used gastric biopsy specimens to detect these point mutations. METHODS: The frequency of primary clarithromycin-resistant H. pylori was examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Eighty-two strains (42 isolated from patients with gastric cancer and 40 isolated from patients with chronic gastritis) were examined. Two biopsy specimens obtained from patients in whom eradication therapy including clarithromycin had failed were also studied. RESULTS: Either A2143G or A2144G point mutation was detected in 90% of clarithromycin-resistant H. pylori strains. Eight out of 82 strains (9.8%) had either A2143G or A2144G point mutation. Only one out of 42 strains in patients with gastric cancer had A2143G mutation, whereas five strains had A2144G and two had A2143G mutations in 40 strains isolated from control subjects. The proportion was significantly lower in patients with early gastric cancer (P < 0.05). This PCR-RFLP was also applicable for DNA samples extracted from biopsy specimens and infection of clarithromycin-resistant H. pylori was observed. CONCLUSION: The results suggest that the point mutation in the 23S rRNA gene is commonly seen in clarithromycin-resistant H. pylori and it contributes to the treatment failure in Japan. The PCR-RFLP system is a sensitive method by which to diagnose H. pylori infection as well as a simple method for detecting clarithromycin resistance without bacterial culture.