控制正常细胞对肺癌细胞P^16基因缺失的污染的方法的建…

目的 建立一种能有效提高从混有正常细胞的肿瘤组织中检测Ｐ＾１６基因纯合缺失的灵敏度的方法，以及探讨该基因的状态与肺癌临床病理的关系。方法 将野生型ＤＮＡ与Ｐ＾１６基因纯合缺失的ＤＮＡ按一定比例混合，并将其有限稀释，以不同量的ＤＮＡ为模板，用聚合酶链反应（ＰＣＲ）扩增Ｐ＾１６基因第二外显子及内对照基因片断，在此基础上，以特定量肺癌ＤＮＡ为模板，用相同的ＰＣＲ条件对上述片断进行扩增，对扩增出的第二外显     

原发性非小细胞肺癌的P~(16)基因缺失及其临床意义

应用聚合酶链反应（ＰＣＲ）技术对３１例非小细胞肺癌（ＮＳＣＬＣ）标本进行了Ｐ１６基因纯合性缺失检测,以了解Ｐ１６基因缺失与ＮＳＣＬＣ组织病理类型、临床分期的关系。１　资料与方法１１　标本来源　３１例ＮＳＣＬＣ标本为山东省立医院、山东省肿瘤防治院、山东医科大学附属医院住院患者的手术切除标本,并经病理证实。获取标本后１小时内用液氮冷却,保存在－７０℃冰箱中备用。应用常规方法提取肿瘤细胞基因组ＤＮＡ,紫外分光光度计测定ＤＮＡ浓度和纯度,取少量ＤＮＡ以备ＰＣＲ扩增。１２　根据已知Ｐ１６基因序列,在第…     

原发性肝癌中p16基因甲基化的研究

应用ＰＣＲ甲基化分析法检测２５例原发性肝癌（ＨＣＣ）院标本及相应的癌旁组织中Ｐ１６基因甲基化情况。１资料与方法：２５例ＨＣＣ及相应的癌旁组织标本均为手术切除标本,置－８０℃冻存。参照《分子克隆》方法提取基因组Ｄ＼Ａ,测定Ａ２６０／Ａ２８０比值在１．８～２．０之间。分析ｐ１６基因第１外显子的甲基化情况、采用在ｐ１６第１外显子序列内有酶切位点的甲基敏感的限制性内切酶Ｓｍａｌ作为基因组ＤＮＡ的消化酶,以不含Ｓｍａｌ位点的染色体９Ｐ标记点Ｄ９Ｓ１６２作为内对照。ＰＣＲ扩增所用引物序列及合成的片断大小为：…     

155例肺癌中P53基因突变的研究

目的 探讨Ｐ５３基因突变与人肺癌临床病理特征及预后、复发转移之间的关系。方法 应用聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）检测１５５例原发肺癌手术标本中Ｐ５３基因５－８外显子的突变。结果 非小细胞肺癌（ＮＳＣＬＣ）中Ｐ５３基因突变率为５３．８％（７８／１４５），小细胞肺癌（ＳＣＬＣ）为８０％（８／１０）。近一半的临床早期ＮＳＣＬＣ患者有Ｐ５３基因突变（４２．５％），晚期Ｐ５３基因突变率     

有关p16基因在胃癌和结肠癌中缺失状态的研究

为了研究在多种原发性消化道肿瘤中有无存在Ｐ１６５基因缺失的状态，采用ＰＣＲ、ＳＳＣＰ分析及ＤＮＡ序列测定技术专门研究了１２例胃癌、结肠癌患者的病理标本。结果表明：（１）Ｐ１６基因缺失的检出率随不同的肿瘤组织类型而异；（２）分化程度低的消化道肿瘤Ｐ１６基因缺失的检出率较高。提示Ｐ１６基因的缺失可能与某些原发性恶性消化道肿瘤的发生或发展有关。     

肺癌组织频繁出现FHIT基因转录本的缺失

目的研究肺癌组织中脆性组氨酸三位体（ＦＨＩＴ）基因的改变。方法采用逆转录聚合酶链反应（ＲＴＰＣＲ）和逆转录聚合酶链反应单链构象多态性（ＲＴＰＣＲＳＳＣＰ）方法分析４７例肺癌组织和其中１６例相应的转移性肺门淋巴结。结果６８％（３２／４７）肺癌原发灶（包括２例肺鳞状细胞原位癌）和９４％（１５／１６）转移性肺门淋巴结出现ＦＨＩＴ转录本缺失,二者统计学差异有显著性（Ｐ＜０．０５）。ＦＨＩＴ基因转录本缺失主要发生于编码区。ＲＴＰＣＲＳＳＣＰ分析未发现突变。结论（１）在肺癌组织中ＦＨＩＴ基因转录本缺失是频发事件,且可能为早期事件;（２）ＦＨＩＴ基因突变可能是少见事件;（３）转移性肺门淋巴结ＦＨＩＴ基因转录本缺失频率与肺癌原发灶相比差异有显著性,提示具有ＦＨＩＴ转录本缺失的肺癌细胞具有更明显的恶性表型和恶性行为     

PCR—单链构象多态性分析在中国大陆日本血吸虫品系研 …

聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）是一种将ＰＣＲ扩增的ＤＮＡ片断变性后在中性聚丙烯酰胺凝胶中电泳，检测点突变和ＤＮＡ多态性的有效方法，本实验采用ＰＣＲ－ＳＳＣＰ分析对我国江苏，云南，湖南及湖北４省的日本血吸虫染色体ＤＮＡ中１个２８０ｂｐ的ｒＤＮＡ片断进行了比较分析，结果显示，４地血吸虫２８０ｂｐ的ｒＤＮＡ片断有一定的多态性和碱基序列的差异，其中江苏和云南两地样品带型相似，而湖南和     

肺癌患者组织中p15、p16纯合缺失

目的：探讨ｐ１５、ｐ１６基因在肺癌组织中屯合缺失情况。方法对５９例肺癌组织和１４例肺癌良性组织,应用ＰＣＲ法检测Ｐ１５、Ｐ１６基因纯合缺失情况。结果在肺癌组织中Ｐ１５、Ｐ１６基因改变频率分别为１１．９％、２３．８％。而在肺部良性组织中只有ＰＨ基因１例阳性,结论Ｐ１６基因在肺癌组织中改变率相对较高;Ｐ１５、Ｐ１６基因在肺癌的研究中可能具有一定的价值。     

PCR-单链构象多态性分析在中国大陆日本血吸虫品系研究中的应用

聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）是一种将ＰＣＲ扩增的ＤＮＡ片断变性后在中性聚丙烯酰胺凝胶中电泳，检测点突变和ＤＮＡ多态性的有效方法。本实验采用ＰＣＲ－ＳＳＣＰ分析对我国江苏、云南、湖南及湖北４省的日本血吸虫染色体ＤＮＡ中１个２８０ｂｐ的ｒＤＮＡ片断进行了比较分析，结果显示：４地血吸虫２８０ｂｐ的ｒＤＮＡ片断有一定的多态性和碱基序列的差异，其中江苏和云南两地样品带型相似，而湖南和湖北两地样品带型相似     

丙型肝炎病毒聚合酶链反应直接序列分析方法的建立及应用(英文)

目的探讨快速完成丙型肝炎病毒ｃＤＮＡ（ＨＣＶｃＤＮＡ）序列分析的方法．方法将聚合酶链反应（ＰＣＲ）扩增与核酸序列分析技术相结合，以ＰＣＲ产物为测序模板，以ｒ３２ＰＡＴＰ标记ＰＣＲ扩增引物为测序引物，用ｔａｑＤＮＡ聚合酶直接测序．结果以琼脂糖或聚丙烯酰胺凝胶电泳回收ＰＣＲ产物制备测序模板可获得最佳测序效果．在需要测定大量标本时，为进一步简化操作步骤，可降低套式ＰＣＲ中第一次扩增反应的ｄＮＴＰ与引物浓度，以第一次ＰＣＲ扩增产物为模板，也可得到较好的测序结果。ＰＥＧ沉淀回收法不能去除非特异性ＰＣＲ产物，对ＰＣＲ直接测序有一定影响．采用此方法首次在一株ＨＣＶＮＳ５ｂ区发现一个新的缺失突变．结论ＰＣＲ直接序列分析是一种简便，快速，经济有效的核酸序列分析方法，适用于丙型肝炎病毒基因组的分析研究．     

NSCLC患者呼出气冷凝液中p16基因突变的研究

目的 研究非小细胞肺癌患者(NSCLC)呼出气冷凝液(EBC)中p16基因突变的临床意义.方法 收集30例NSCLC患者和20例体检健康者的EBC标本,提取EBC中的DNA,对β-actin基因扩增阳性的EBC标本进行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例肺癌患者的EBC中,有26例β-actin基因片段扩增阳性,26例中有9例检出p16基因突变,突变率为34.6％.②9例肺癌患者发生p16基因突变的外显子,1号外显子3例,2号外显子5例,3号外显子1例.③26例肺癌患者EBC中β-actin基因片段扩增阳性中,Ⅰ期12例,p16基因突变3例,突变率25％;Ⅱ期7例,p16基因突变2例,突变率28.6％;Ⅲ期7例,p16基因突变4例,突变率57.1％.鳞癌14例,p16基因突变6例,突变率42.9％;腺癌11例,p16基因突变3例,突变率27.3％.结论 NSCLC患者EBC中可以检测到p16基因突变,特异性高,EBC中p16基因检测为肺癌研究提供新方法.     

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.     

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.     

Expression, deleton and mutation of p16 gene in human gastric cancer

AIM To investigate the relationship between the expression of p16 gene and the gastric carcinogenesis,depth of invasion and lymph node metastases, and to evaluate the deletion and mutation of exon 2 in p16 gene in gastric carcinoma. METHODS The expression of P16 protein was examined by streptavidin-peroxidase conjugated method (S-P); the deletion and mutation of p16 gene were respectively examined by polymerase chain reaction (PCR) and polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) in gastric carcinoma. RESULTS Expression of P16 protein was detected in 96.25% (77/80) of the normal gastric mucosa, in 92.00% (45/50) of the dysplastic gastric mucosa and in 47.54% (58/122) of the gastric carcinoma. The positive rate of P16 protein expression in gastric carcinoma was significantly lower than that in normal gastric mucosa and dysplastic gastric mucosa (P＜0.05). The positive rate of P16 protein expression in mucoid carcinoma 10.00% (1/ 10) was significantly lower than that in poorly differentiated carcinoma 51.22% ( 21/ 41 ),undifferentiated carcinoma 57.69% (15/26) and signet ring cell carcinoma 62.50% (10/ 16) (P＜0.05). The positive rate of p16 protein in 30 cases paired primary and lymph node metastatic gastric carcinoma: There was 46.67% (14/30) in primary gastric carcinoma, 16.67% (5/30) in lymph node metastatic gastric carcinoma. The positive rate of lymph node metastatic carcinoma was significantly lower than that of primary carcinoma (P＜0.05). There was of p16 gene mutation in exon 2, but 5 cases displayed deletion of p16 gene in exon 2 in the 25 primary gastric carcinomas. CONCLUSIONS The expression loss of P16 protein related to the gastric carcinogenesis, gastric carcinoma histopathological subtypes and lymph metastasis. The mutation of p16 gene in exon 2 may not be involved in gastric carcinogenesis. But the deletion of p16 gene in exon 2 may be involved in gastric carcinogenesis.     

胰腺癌抑癌基因P16、DPC4寡核苷酸芯片点突变及缺失的应用

目的建立P16、DPC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P16、DPC基因突变和缺失。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例胰腺组织标本中有7例检测出存在着P16基因的改变，4例标本为点突变，2例标本为缺失型改变，还有1例患者存在着野生型与缺失型同时存在的杂和状态。有7例标本发生了D即4基因的改变，有6例突变，1例为缺失，基因异常比例为43.75％。结论P16、DPCA基因芯片可同时检测胰腺癌多个突变位点基因。     

Expression, deletion [was deleton] and mutation of p16 gene in human gastric cancer

AIM: To investigate the relationship between the expression of p16 gene and the gastric carcinogenesis, depth of invasion and lymph node metastases, and to evaluate the deletion and mutation of exon 2 in p16 gene in gastric carcinoma. METHODS: The expression of p16 protein was examined by streptavidin-peroxidase conjugated method (S-P);the deletion and mutation of p16 gene were respectively examined by polymerase chain reaction (PCR) and polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP) in gastric carcinoma. RESULTS: Expression of p16 protein was detected in 96.25% (77/80) of the normal gastric mucosa, in 92.00% (45/50) of the dysplastic gastric mucosa and in 47.54% (58/122) of the gastric carcinoma. The positive rate of p16 protein expression in gastric carcinoma was significantly lower than that in normal gastric mucosa and dysplastic gastric mucosa (P < 0.05). The positive rate of p16 protein expression in mucoid carcinoma 10.00% (1/10) was significantly lower than that in poorly differentiated carcinoma 51.22% (21/41), undifferentiated carcinoma 57.69% (15/26) and signet ring cell carcinoma 62.50% (10/16) (P < 0.05). The positive rate of p16 protein in 30 cases paired primary and lymph node metastatic gastric carcinoma: There was 46.67% (14/30) in primary gastric carcinoma, 16.67% (5/30) in lymph node metastatic gastric carcinoma. The positive rate of lymph node metastatic carcinoma was significantly lower than that of primary carcinoma (P < 0.05). There was of p16 gene mutation in exon 2, but 5 cases displayed deletion of p16 gene in exon 2 in the 25 primary gastric carcinomas. CONCLUSIONS: The expression loss of p16 protein related to the gastric carcinogenesis, gastric carcinoma histopathological subtypes and lymph metastasis. The mutation of p16 gene in exon 2 may not be involved in gastric carcinogenesis. But the deletion of p16 gene in exon 2 may be involved in gastric carcinogenesis.     

老年非小细胞肺癌患者表皮生长因子受体基因19-21外显子突变研究

目的 研究老年非小细胞肺癌(NSCLC)患者表皮生长因子受体(epidermal growth factor receptor,EGFR)基因酪氨酸激酶区19-21外显子的突变状态,并对其临床特征进行初步分析.方法 选取46例病理确诊的老年非小细胞肺癌患者,提取肺癌组织或胸腔积液及心包积液肿瘤细胞基因组DNA,通过巢式PCR基因扩增和直接测序的方法 .分析19、20、21外显子的突变情况,并与其临床特征及应用酪氨酸激酶抑制剂(TKI)疗效的关系进行了初步分析. 结果 46例中,26例检测出带有基因突变(56.5%),其中非沉默突变者为19例(41.3%).19号外显子突变6例(13.0%),20号外显子突变13例(28.2%),21号外显子突变14例(30.4%).其中7例带有2处突变,其余均为单处突变.不吸烟者的突变发生率显著高于吸烟者(P＜0.01).应用酪氨酸激酶抑制剂(TKI)临床获益的患者带有EGFR 19、20、21外显子的基因突变的几率显著高于未获益者(P＜0.05).60～69岁与70～85岁年龄组基因突变状态差异无统计学意义. 结论 老年NSCLC患者EGFR基因酪氨酸激酶区19-21外显子突变特征与肺癌患者总体类似,与年龄关系不大,老年NSCLC患者同样可以通过基因检测获得TKI治疗预测信息.     

抑癌基因P16在肺鳞癌和肺腺癌中的表达及临床意义

目的 探讨p16基因产物在肺鳞癌和肺腺癌中的表达及其意义。方法 本组56例原发性非小细胞肺癌，其中鳞癌37例，腺癌19例。用免疫组织化学PCR法检测患者肺癌新鲜标本p16蛋白表达水平。结果 56例肺癌标本中p16蛋白阳性表达率为58.9%（33／56），伴有淋巴结转移者其阳性表达率41.4%（12／29）显著低于无淋巴结转移者（P16阳性表达率为77.8%（21／27），P＜0.01）。P16蛋白阴性表达者的1年、3年生存率分别为59.7%、44.1%，显著低于p16蛋白阳性表达者85.2%、71.8%。结论 p16蛋白表达与肺鳞癌和肺腺癌的组织类型，淋巴结转移及预后有关。p16蛋白状态可作为判断肺癌预后的指标之一。