快速, 高敏感度检测Ki—ras基因突变方法的建立及其在检测大肠癌中的应用

建立一种检测Ｋｉ－ｒａｓ基因突变的快速、高敏感度的方法，并将其应用于大肠癌的检测中，应用引物导限制富集突变等位基因检测法（ＰＣＲ＿ＰＩＲＥＭＡ，Ｐｒｉｍｅｒ Ｉｎｔｒｏｄｕｃｅｄ Ｒｅｓｔｒｉｃｔｉｏｎ ｗｉｔｈ Ｅｎｒｉｃｈｍｅｎｔ ｏｆ Ｍｕｔａｎｔ ＡｌｌｅｌｅＡｓｓａｙ）的敏感度达１０＾－５－１０＾－６，并在３０例大肠癌样品中检测出了８例Ｋｉ－ｒａｓ基因１２密码子突变的样本，突变率达２６．     

ras基因与人类肺癌

本文对近几年ｒａｓ基因突变与肺癌的关系研究做了回顾，发现Ｋ－ｒａｓ基因点突变与肺癌的发生有一定的关系。     

接触苯工人白细胞癌基因激活的研究

应用ＰＣＲ－Ｂｌｏｔ和Ｄｏｔ－Ｂｌｏｔ技术检测了１０３名接触苯工人和７０名对照组工人外周血白细胞ｃ－Ｋｉ－ｒａｓ２基因和ｃ－ｍｙｃ基因。结果表明，１０３名接苯者中有４例其外周血白细胞检出ｃ－Ｋｉ－ｒａｓ２基因１２位点Ｇ→Ｔ突变，其接苯工龄在６～１０年，而７０名对照者未检出该突变；ｃ－Ｋｉ－ｒａｓ２基因１２位点（Ｇ→Ａ突变在两组工人中均未检出阳性；ｃ－ｍｙｃ基因亦未在两组工人中检出扩增现象。结果提示，ｃ－Ｋｉ－ｒａｓ２基因１２位点Ｇ→Ｔ突变与慢性接触苯有关，有可能是苯白血病发生的分子机理。     

RAS基因及表达产物P_(21)在妇科肿瘤中的研究进展

Ｐ２１系由原癌基因ＲＡＳ家族（Ｋ－ｒａｓ、Ｈ－ｒａｓ、Ｎ－ｒａｓ）所编码的一种蛋白质，其分子量为２１ＫＤ，是细胞周期中重要的调控因子。当ＲＡＳ基因发生突变时，可以导致原癌基因激活，使Ｐ２１异常表达，参与肿瘤的发生和发展。本文就ＲＡＳ基因及其表达产物Ｐ２１在妇科肿瘤中的研究进展作一综述。     

RAS基因及表达产物P21在妇科肿瘤中的研究进展

Ｐ２１系由原癌基因ＲＡＳ家族（Ｋ－ｒａｓ，Ｈ－ｒａｓ，Ｎ－ｒａｓ）所编码的一种蛋白质，其分子量为２１ＫＤ，是细胞周期中重要的调控因子，当ＲＡＳ基因发生突变时，可以导致原癌基因激活，使Ｐ２１异常表达，参与肿瘤的发生和发展，本文就ＲＡＳ基因及其表达产物Ｐ２１在妇科肿瘤中的研究进展作了一综述。     

苯作业工人白细胞K_i-ras基因点突变分子检测初探

苯作业工人白细胞Ｋ_ｉ－ｒａｓ基因点突变分子检测初探夏昭林，金锡鹏，陆培廉，顾学箕，夏俊杰，丁晓华（上海医科大学公共卫生学院，２０００３２）人类ｒａｓ基因包括Ｈａ－ｒａｓ、Ｋｉ－ｒａｓ、和Ｎ－ｒａｓ三个成员，ｒａｓ基因常通过点突变尤其第１２位密码子突?..     

青石棉与苯并（a）芘联合诱发大鼠肺癌Ki－ras癌基因点突变

用青石棉＋苯并（ａ）芘碘油混悬液对大鼠气管注入染尘，取染尘后１８０和２７０天剖检的肺组织标本２５例。提取标本中ＤＮＡ，用人工合成的寡核苷酸引物进行ＰＣＲ体外扩增，再用γ－３２ＰＡＴＰ标记正常和两种突变Ｋｉ－ｒａｓ１２位探针，杂交，放射自显影。结果表明，正常对照组７例标本Ｋｉ－ｒａｓ１２位点突变为阴性，青石棉＋苯并（ａ）芘组１８例标本中，３例Ｋｉ－ｒａｓ１２位点突变为阳性，其中２例由ＧＧＴ（Ｇｌｙ）突变为ＡＧＴ（Ｓｅｒ），１例由ＧＧＴ（Ｇｌｙ）突变为ＴＧＴ（Ｃｙｓ），并对突变原因进行了探讨。     

PCR-SSCP-DNA序列分析法检测人肺癌中K-ras基因突变

应用聚合酶链式反应（ＰＣＲ）－单链构象多态性（ＳＳＣＰ）－ＤＮＡ序列分析法，对４０例肺癌样品中Ｋ－ｒａｓ基因突变进行检测。ＰＣＲ－ＳＳＣＰ银染分析，突变检出率为３０％（１２／４０），发现于肺腺癌的Ｋ－ｒａｓ基因突变率为４４％（１２／２７）；ＤＮＡ序列分析，１２例筛选突变阳性样品中的１１例检出了突变位点，９０％的突变发生于１２密码子，其突变谱以Ｇ→Ｔ颠换（占４０％）和Ｇ→Ａ转换（占６０％）为主。本研究结果显示ＳＳＣＰ银染分析对大量样品进行阳性筛选有很好的应用价值，ＰＣＲ－ＳＳＣＰ－ＤＮＡ序列分析法是检测基因突变的有效手段。     

苯作业工人白细胞Ki─ras癌基因点突变分子检测初探

本文应用ＰＣＲ技术和寡聚核苷酸探针检测苯作业工人外周血白细胞中Ｋｉ─ｒａｓ癌基因第１２位密码子点突变。４１名接触苯工人和４１名对照工人中只有１例接苯工人发生ＧＧＴ→ＴＧＴ（Ｇｌｙ→Ｃｙｓ）突变，该女工苯作业工龄６年，连续三次（均间隔三个月）白细胞计数均呈低下状态，但其他血液学指标未见明显异常。初步探讨了Ｋｉ─ｒａｓ基因点突变检测作为苯白血病的生物标志和职业人群早期监护指标的可能性。     

青石棉诱发大鼠胸膜间皮瘤

本文采用右胸腔内注入染尘法，对大鼠染青石棉尘４０ｍｇ。收集１９例胸膜间皮瘤标本，提取标本中ＤＮＡ，用人工合成的寡核苷酸引物行ＰＣＲ体外扩增，再用ｒ－（３２）ＰＡＴＰ标记正常和两种空变Ｋｉ－ｒａｓ１２位探针，杂交，放射自显影。结果表明，７例正常对照组标本Ｋｉ－ｒａｓ１２位点突变为阴性；１９例间皮瘤标本中，２例突变阳性，其中１例由ＧＧＴ（Ｇｌｙ）突变为ＡＧＴ（Ｓｅｒ），１例由ＧＧＴ（Ｇｌｙ）突变为ＴＧＴ（Ｃｙｓ）。     

84例晚期结直肠癌K-ras基因突变情况研究

目的观察K-ras基因在晚期结直肠癌原发灶及对应转移灶中的突变情况。方法采用DNA直接测序法检测84例晚期结直肠癌患者原发灶及对应转移灶K-ras基因的突变情况。结果 26例原发灶及对应转移灶K-ras基因均为突变型;52例原发灶及对应转移灶K-ras基因均为野生型。原发灶及转移灶两者的一致率为92.86%,无统计学差异(p=1.00)。两者不一致的情况有6例(7.14%)。结论晚期结直肠癌原发灶和对应转移灶中K-ras基因的突变具高度一致性,可作为临床选用分子靶向药物的依据。     

石棉及电焊烟尘相关肺癌组织K-ras基因突变的研究

为了解石棉及电焊烟尘相关肺癌的Ｋ－ｒａｓ基因突变情况,并与非职业肺癌的Ｋｒａｓ基因突变特点比较,进一步探讨石棉及电焊烟尘的致癌机理。收集８例非职业肺癌组织、９例石棉相关肺癌组织及４例电焊烟片段长度多态性分析,单链构象多态性分析及ＤＮＡ真接测序分析。     

Alcohol and the risk of colon and rectal cancer with mutations in the K-ras gene.

The first metabolite of alcohol, acetaldehyde, may trigger replication errors and mutations in DNA, which may predispose to developing colorectal cancer (CRC). In a prospective study on colon and rectal cancer, we investigated the following hypotheses: alcohol consumption is associated with an increased risk of mutations in the K-ras oncogene, and beer consumption is associated with an increased risk of G-->A mutations in this gene. Therefore, we studied the associations between consumption of alcohol and alcoholic beverages and the risk of CRC without and with specific K-ras gene mutations. In 1986, 120,852 men and women, aged 55-69 years, completed a questionnaire on risk factors for cancer. The case-cohort approach was used for data processing and analyses. After 7.3 years of follow-up, excluding the first 2.3 years, complete data from 4,076 subcohort members, 428 colon and 150 rectal cancer patients, were available for data analyses. Incidence rate ratios (RRs) and corresponding 95% confidence intervals (95% CIs) were estimated using Cox proportional hazards models. Compared to abstaining, a total alcohol consumption of 30.0 g/day and more was associated with the risk of colon and rectal cancer with and without a K-ras mutation in both men and women. Independent from alcohol intake, liquor consumption when compared to nonliquor consumption was associated with an increased risk of rectal cancer with a wild type K-ras in men (RR: 2.25, 95% CI: 1.0-5.0). Beer consumption was not clearly associated with the risk of colon and rectal tumors harboring G-->A mutations in the K-ras gene in men. This association could not be assessed in women because of sparse beer consumption. In conclusion, alcohol does not seem to be involved in predisposing to CRC through mutations in the K-ras gene, and specifically beer consumption is not associated with colon and rectal tumors harboring a G-->A mutation.     

肺癌患者支气管肺泡灌洗液中K-ras基因第1外显子点突变与吸烟的关系

[目的 ]探讨支气管肺泡灌洗液 (BALF)细胞中K ras原癌基因点突变与吸烟的关系及其在肺癌早期诊断中的意义。 [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) ,检测了 3 7例吸烟肺癌患者、2 5例非吸烟肺癌患者以及 2 0例肺良性疾病支气管肺泡灌洗液细胞中K ras基因第 12位密码子突变的情况。 [结果 ]肺癌患者K ras基因突变率为 3 5 5 % (2 2 / 62 ) ,肺良性疾病中未发现有K ras基因突变者。突变的发生与患者的年龄及吸烟史有关 ,与性别、TNM分期无关 ;另外 ,重度吸烟者突变率 (68 8% )高于轻度 (2 5 0 % )及中度吸烟者 (3 0 8% ) ,(P <0 0 5 ) ,更明显地高于非吸烟者 (2 0 0 % ) ,(P <0 0 1)。 [结论 ]①吸烟可能是导致K ras基因突变的重要因素之一 ;②BALF细胞中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     

Association between coffee drinking and K-ras mutations in exocrine pancreatic cancer. PANKRAS II Study Group

STUDY OBJECTIVE: To analyse the relation between coffee consumption and mutations in the K-ras gene in exocrine pancreatic cancer. DESIGN: Case-case study. Consumption of coffee among cases with the activating mutation in the K-ras gene was compared with that of cases without the mutation. SETTING AND PATIENTS: All cases of pancreatic cancer newly diagnosed at five hospitals in Spain during three years were included in the PANKRAS II Study (n = 185, of whom 121 whose tissue was available for molecular analysis are the object of the present report). Over 88% were personally interviewed in hospital. DNA was amplified from paraffin wax embedded tissues, and mutations in codon 12 of K-ras were detected by the artificial RFLP technique. MAIN RESULTS: Mutations were found in tumours from 94 of 121 patients (77.7%). Mutations were more common among regular coffee drinkers than among non-regular coffee drinkers (82.0% v 55.6%, p = 0.018, n = 107). The odds ratio adjusted by age, sex, smoking and alcohol drinking was 5.41 (95% CI 1.64, 17.78). The weekly intake of coffee was significantly higher among patients with a mutated tumour (mean of 14.5 cups/week v 8.8 among patients with a wild type tumour, p < 0.05). With respect to non-regular coffee drinkers, the odds ratio of a mutated tumour adjusted by age, sex, smoking and alcohol drinking was 3.26 for drinkers of 2-7 cups/week, 5.77 for drinkers of 8-14 cups/week and 9.99 for drinkers of > or = 15 cups/week (p < 0.01, test for trend). CONCLUSIONS: Pancreatic cancer cases without activating mutations in the K-ras gene had drank significantly less coffee than cases with a mutation, with a significant dose response relation: the less they drank, the less likely their tumours were to harbour a mutation. In exocrine pancreatic cancer the K-ras gene may be activated less often among non-regular coffee drinkers than among regular drinkers. Caffeine, other coffee compounds or other factors with which coffee drinking is associated may modulate K-ras activation.     

利用激光捕获显微分离技术和突变分析法检测肺癌病人痰细胞的K—ras和p53基因突变

[目的]探索激光捕获显微分离技术在检测肺癌病人K-ras和p53基因突变的应用。[方法]以3种不同的基因突变分析法对20例患者的痰细胞进行基因突变分析：①以PCR＋DGGE方法分析K—ras基因突变，以PCR＋SSCP方法分析p53基因突变；②以PCR十MAE＋DGGE方法分析K—ras基因突变；以PCR＋MAE＋SSCP方法分析p53基因突变；③以激光捕获显微分离技术和突变分析法检测肺癌病人痰细胞的K—ras和p53基因突变。[结果]在20例患者中，以现有的方法仅发现有15％（3／20）的基因突变，而激光捕获显微分离技术则发现有60％（12／20）的基因突变。[结论]激光捕获显微分离技术和突变分析法能高效灵敏检测肺癌病人痰细胞的K—ras和p53基因突变。     

K-ras基因状况预测转移性结直肠癌药物治疗后生存期

目的旨在通过前瞻性检测转移性结直肠癌的K-ras基因表达状况,观察比较化疗单用或联合靶向治疗的长期疗效,为临床医生提供可靠预测指标。方法初治的转移性结直肠癌62例,一线化疗前全部检测了癌组织K-ras基因,62例癌组织K-ras基因进行检测,突变率为30.6%(19/62),其中G12占89.5%(17/19),G13占10.5%(2/19);43例K-ras野生型者分两组进行姑息化疗,包括单纯化疗组及化疗联合靶向治疗组,治疗期间密切随访,统计疗效及毒性并进行统计学分析。结果 K-ras突变者中位PFS和OS分别为4.7月和14.5月,野生型者中位PFS和OS分别为8.5月和24.0月,差异均有统计学意义(Log-rank检验P﹤0.05);10名K-ras野生型者在化疗基础上加用靶向治疗(西妥昔单抗),其中位生存期36.5月,显著高于未用靶向治疗患者的18.0月(Log-rank检验P﹤0.01)。结论癌组织K-ras基因突变者生存期明显短于K-ras基因野生型者,表明K-ras基因突变可作为本组转移性结直肠癌的有效预后预测指标;化疗联合靶向治疗转移性结直肠癌,可有效改善K-ras基因野生型者的PFS和OS,值得推广应用。     

K-ras与非小细胞肺癌的研究进展

Ras蛋白(P21ras)属于鸟苷酸三磷酸酶(GTPase)家族成员,是细胞增殖、分化、分裂和凋亡的重要调节者。已经发现有20%～30%的非小细胞肺癌(non-smallcelllungcancer,NSCLC)存在K-ras基因突变,认为其在恶性发生中扮演了重要角色。20多年来许多研究认为K-ras基因突变可作为一个不良的预后标志,同样也有很多研究没能证实这一观点。研究进展的阻碍与相对小规模的研究,不同的分子分析方法,和组织来源的异源性、组织的不同亚型、分期、治疗和生存标准有关。最近发现NSCLC患者使用辅助治疗或者使用表皮生长因子受体(EGFR)抑制剂都对病程有不同影响,K-ras基因是EGFR途径下游的一个重要信号分子,因此认为K-ras基因也是NSCLC一个重要的生物标志,值得进一步深入研究。     

Prognostic value of the presence of the mutation of the codons 12, 13 and 61 in K-ras oncogene in colorectal cancer]

Despite of extensive and intensive investigations, the predictive and prognostic value of c-K-ras mutation is not unequivocal. There has been reported about investigation the occurrence of mutations in the 88 colorectal cancer patient's specimen using polymerase chain reaction. Age: 61.9 years (27-80), gender 8 male, 42 female. Dukes' stages: 43 at the B, 35 at C, 10 at D. Primary of tumour: 52 colon, 36 rectal adenocarcinoma. Mutation out of one of the three ras-codons was detectable in the 54 cases, more frequently at the stage Dukes' C (p < 0.05). The ras-mutation concerned to more elevated death-rate in the stages Dukes' B and C (p < 0.01). Mean survival time to progression was significantly longer at the stage Dukes' B if mutation had not been detected (p < 0.01). The occurrence of the rate of genetic alteration was significantly more frequent at tumours of right-side colon, than left side (p < 0.02) or rectum (p < 0.05) one's. However, at the age of 41-50 years it was significantly more presented at the cases of rectal cancer (p < 0.01). At the age of 51-60 years mutations were detected among men at higher rate (p < 0.05). The cases of local recurrences concerned by mutation at the codon of 13 (p < 0.05). Occurrence of ras-oncogene is the sign of extremely malignant potential of tumour. This fact manifested itself in the time to progression and mean survival time of patients at same clinical or pathological stage. The higher frequency of genetic alterations at the proximal colon may be the reason of more unfavourable prognosis of the disease localised to this site. Reconstructing the molecular events, the presence of ras mutation can serve as a basis for prognosis of the disease and permit of potentially individualised therapeutic intervention.