巢式甲基化特异性PCR 检测食管癌病人血清中p16 基因启动子区过甲基化

 目的 检测食管鳞状细胞癌（squamous cell carcinoma，SCC）患者外周血血清中p16基因启动子区的甲基化状态，探讨p16基因启动子的过甲基化在食管鳞状细胞癌筛查及早期诊断中的意义。方法 利用巢式甲基化特异性PCR（nMSP）法检测食管鳞状细胞癌患者外周血血清与正常人血清中p16基因启动子的甲基化状态，并与普通甲基化特异性PCR（MSP）法进行了比较。结果 56例SCC血清样品中nMSP法发现34例p16基因启动子的过甲基化，MSP法只检出15例，而22例正常人血清中都未检测到p16基因启动子的过甲基化。测序结果进一步验证了方法的可靠性。结论 利用巢式MSP（nMSP）法检测外周血血清中p16基因启动子的甲基化，可为食管癌的筛查、早期诊断及预后判断提供有价值的信息。     

基于p73和DAPK基因异常甲基化模式的白血病肿瘤标志物研究

目的 肿瘤抑制基因异常甲基化状态与白血病发生关系密切,发展白血病甲基化标志物成为研究热点.但白血病类型复杂,利用单个基因甲基化模式作为肿瘤标志物具有一定局限性.本研究通过探讨基于肿瘤抑制基因p73和死亡相关蛋白激酶(death-associated protein kinase gene,DAPK)异常甲基化模式对白血病诊断分型的价值和意义,寻找一种联合多基因异常甲基化模式作为白血病肿瘤标志物的方法.方法 应用亚硫酸氢盐测序法(bisulfite sequencing PCR,BSP),分析正常人白细胞、单核细胞系白血病细胞株U937、粒细胞系HL-60及淋巴细胞系Jurkat基因组中,p73和DAPK基因启动子区CpG岛甲基化状态,将测序结果汇集到Office Excel 2010文件中,并计算各CpG位点甲基化率,绘制2个基因在4种细胞来源基因组中的CpG岛甲基化模式图,从而筛选特异甲基化位点组合.通过甲基化特异性PCR法(methylation specific PCR,MSP),在白血病细胞株、30例正常对照和104例白血病惠者外周血标本中,验证基因异常甲基化模式的诊断效能.结果 成功测序约107个含BSP产物转化质粒的菌株,并绘制出p73和DAPK基因CpG岛甲基化模式图.正常细胞基因组两者去甲基化程度远高于白血病细胞株.p73基因在正常细胞与白血病细胞株中,甲基化状态完全相反.DAPK基因在HL-60中与其他白血病细胞株甲基化状态存在明显差异.p73基因MSP甲基化检测可以鉴别正常细胞和白血病细胞,在临床白血病标本诊断中的灵敏度、特异度和准确度分别是21.5％、100.0％和43.1％,DAPK基因甲基化检测可以鉴别HL-60与其他白血病细胞株,其诊断急性非淋巴细胞白血病的灵敏度、特异度和准确性分别是59.1％、100.0％和77.2％.结论 p73和DAPK基因启动子区CpG岛在不同类型白血病细胞基因组中存在特异的甲基化位点,将其作为白血病初步诊断分型的潜在肿瘤标志物具有一定临床意义,也为今后发现白血病等肿瘤的甲基化标志物提供方法并奠定实验研究基础.     

急性白血病P16基因缺失的意义

目的 探讨周期素依赖性激酶4抑制因子基因（p16）缺失在急性白血病中的意义。方法 采用PCR方法对43例急性淋巴细胞白血病（ALL）,36例急性非淋巴细胞白血病（ANLL）中p16基因的纯合缺失进行了研究。结果 21例ALL存在p16基因纯合缺失,3例ANLL存在p16基因纯合缺失。ALL p16基因缺失率明显高于ANLL（P＜0．05）,p15基因缺失的ALL病人有较高的复发率。结论 ALLp16基因缺失率较高,p16基因缺失之ALL预后不良。     

成人急性白血病p16基因的纯合缺失及甲基化研究

目的　观察成人急性白血病p16基因的纯合缺失及甲基化的情况。方法　用PCR法对成人急性白血病患者骨髓标本进行p16基因第 1、第 2外显子纯合缺失及甲基化检测。结果　 1) 71例成人急性白血病患者中p16基因纯合缺失 2例 ,均为B ALL ,AML及正常对照组p16基因未见纯合缺失 ;2 ) 2 8例ALL中 ,5例发生异常甲基化 ;43例ANLL中 ,有 7例发生了异常甲基化。结论　p16基因缺失、异常甲基化可能与白血病的发生及预后有关     

弥漫大B细胞淋巴瘤SHP-1、p15和SOCS-1基因异常甲基化的研究

目的 探讨弥漫大B细胞淋巴瘤（DLBCL）患者SHP-1 、p15和SOCS-1基因异常甲基化在DLBCL的发生、发展中的意义.方法 采用甲基化特异性聚合酶链反应（PCR）技术研究DLBCL淋巴组织标本中SHP-1、p15和SOCS-1基因启动子区域CpG岛的甲基化状态.结果 50例DLBCL患者中有48例（96％）存在SHP-1基因启动子区甲基化,26例（52％）有p15基因启动子区域甲基化,28例（56％）有SOCS-1基因启动子区的甲基化,而15例对照（淋巴结良性反应性增生）组则无SHP-1、p15和SOCS-1基因的甲基化现象.结论 在DLBCL患者中存在的SHP-1、p15和SOCS-1基因启动子区甲基化现象,初步表明SHP-1、p15和SOCS-1基因甲基化在DLBCL的发生、发展中具有一定作用,并对DLBCL的去甲基化治疗提供了理论依据.     

非霍奇金淋巴瘤p15基因甲基化及去甲基化再表达的研究

目的探索p15基因异常甲基化在非霍奇金淋巴瘤(non-Hodgkin’s lymphomas，NHL)中的分布，去甲基化p15基因的再表达。方法检测32例非霍奇金淋巴瘤石蜡标本p15基因甲基化；用去甲基化因子5-Aza2-cdR诱导p15基因甲基化的淋巴瘤细胞株Raji细胞，RT—PCR方法测定p15基因的表达。结果非霍奇金淋巴瘤p15基因操纵区甲基化的发生率为18．8％(6／32)，高度恶性比低度恶性更易发生甲基化，其发生率分别为27.3％和0％，去甲基化因子5-Aza2-cdR在10^-7～10^-6mol／L诱导的raji细胞p15基因去甲基化再表达。结论p15基因甲基化可能是非霍奇金淋巴瘤发病的原因之一，去甲基化治疗是可行的。     

非霍奇金淋巴瘤p73基因甲基化及去甲基化的研究

目的探讨p73基因异常甲基化在非霍奇金淋巴瘤（non—Hodgkin’s lymphomas,NHL）中的分布,去甲基化p73基因的再表达。方法采用甲基化特异性PCR（Methylation specific polymerase chain reaction,MSP）方法检测22例非霍奇金淋巴瘤p73基因CpG岛甲基化状态。使用反转录PCR（Reverse transoription polymerase chain reaction,RT-PCR）方法检测CdR药物作用前后P73基因mRNA的表达情况。结果非霍奇金淋巴瘤p73基因甲基化的发生率为27.3％（6／22）,高度恶性比低度恶性更易发生甲基化,其发生率分别为42．8％和0％,5-杂氮-2’．脱氧胞嘧啶（5-aza-2’-deoxycytidine,CdR）在4．0～8．0μmol/L时可诱导非霍奇金淋巴瘤细胞p73去甲基化。结论p73基因甲基化可能是非霍奇金淋巴瘤发病的原因之一,去甲基化治疗是可行的。     

B细胞淋巴瘤中促凋亡基因MAPK10异常甲基化的研究

 目的　探讨促凋亡基因MAPK10启动子区CpG岛异常甲基化引起的表达沉默在B细胞淋巴瘤中的意义。方法　提取7个B细胞淋巴瘤细胞株的总RNA和基因组DNA。分别用半定量反转录PCR（RT-PCR）和甲基化特异性PCR（MSP）检测细胞株中MAPK10基因的mRNA表达水平和启动子甲基化状态。用MSP检测了24例弥漫大B细胞淋巴瘤（DLBCL）、15例滤泡性淋巴瘤（FL）和12例淋巴结反应性增生（LRH）中MAPK10甲基化状态。并进一步用亚硫酸氢盐-基因组测序法（BGS）验证了部分细胞株和组织标本中MAPK10的甲基化状态。结果　MAPK10在大部分B细胞淋巴瘤细胞株中存在表达沉默现象,并与其启动子区高甲基化密切相关。MAPK10基因高甲基化在DLBCL和FL中的频率分别为71 %（17／24）及100 %（15／15）,而未在LRH标本中检出（0／12）。BGS检测结果证实了上述结果。结论　MAPK10基因在B细胞淋巴瘤中存在高频甲基化,由其所致的基因沉默可能在B细胞淋巴瘤发生发展中起重要作用。MAPK10基因的肿瘤特异性甲基化可作为一个良好的肿瘤分子诊断标志物。     

多发性骨髓瘤患者p15、p16基因甲基化及其与预后的相关性

 目的　探讨p15、p16 基因的高甲基化与多发性骨髓瘤（MM）的发病和预后之间的关系。方法　采用巢式甲基特异性PCR法（nMSP）检测47例MM患者p15、p16基因的甲基化状态,并对患者的临床资料及预后因素进行分析。结果　47例MM患者p15、p16 基因的甲基化比例分别为59.57 %（28／47）、 57. 45 %（27／47）,两者同时存在甲基化者23 例（48.94 %）。Ⅱ、Ⅲ期MM患者p15 、p16基因甲基化率明显高于Ⅰ期患者。结论　p16 、p15 基因的高甲基化与MM的发病及预后相关。     

成人急性白血病p16基因的纯合缺失及甲基化研究

目的观察成人急性白血病p16基因的纯合缺失及甲基化的情况。方法用PCR法对成人急性白血病患者骨髓标本进行p16基因第1、第2外显子纯合缺失及甲基化检测。结果1)71例成人急性白血病患者中p16基因纯合缺失2例，均为B-ALL，AML及正常对照组p16基因未见纯合缺失；2)28例ALL中，5例发生异常甲基化；43例ANLL中，有7例发生了异常甲基化。结论p16基因缺失、异常甲基化可能与白血病的发生及预后有关。     

丙戊酸钠诱导急性淋巴细胞白血病原代细胞凋亡机制的研究

目的 探讨丙戊酸钠（VPA）对急性淋巴细胞白血病（ALL）原代细胞凋亡现象与去甲基化机制之间的联系.方法 选取10例AIL患者原代细胞,MTT法检测VPA对ALL原代细胞增殖抑制作用,DNA ladder试验观察细胞凋亡,Annexin-V-FITC/PI检测细胞早期凋亡,半巢式甲基化特异PCR检测p15INK4B基因甲基化,反转录PCR方法检测p15基因表达水平.实验数据采用SPSS 16.0统计软件处理.结果 VPA可明显抑制细胞增殖,对原代细胞IC50为1.898 mmol/L;DNA ladder试验显示VPA对原代ALL细胞的凋亡作用明显.Annexin-V-FITC/PI检测1.0、2.0、4.0 mmol/LVPA组的凋亡率分别是（5.80±0.65）％、（48.46±2.49）％、（76.45±2.98）％,与未用药组的（0.44±0.04）％比较差异有统计学意义（P＜0.05）.原代ALL细胞经VPA作用后,p15INK4B甲基化水平明显下降,2.0、4.0 mmol/LVPA组与未用药组相比,p15 mRNA表达水平增强,差异有统计学意义（P＜0.05）.结论 VPA通过使p15INK4B基因去甲基化,促进p15基因表达上调,从而促进原代ALL细胞凋亡.     

Aberrant methylation in promoter-associated CpG islands of multiple genes in relapsed childhood acute lymphoblastic leukemia

Methylation profile was analyzed in nine cases of relapsed childhood acute lymphoblastic leukemia (ALL) for p14, p15, p16, Rb, MGMT, APC, hMLH1, RARbeta, RIZ, DAPK, and FHIT genes by using methylation specific polymerase chain reaction (MSP) analysis. Frequency of methylation in each gene was: MGMT, 56%; RARbeta, 44%; and p16, 22%, respectively. None of the p14, p15, Rb, APC, hMLH1, RIZ, DAPK, and FHIT genes were hypermethylated. Five (56%) of 9 cases showed methylation of at least one gene. All of the samples with hypermethylation in p16 and MGMT gene at relapse, had already acquired the change at the time of initial diagnosis. Interestingly, three of 4 cases with RARbeta gene methylation at relapse did not have methylation of this gene at the time of initial presentation. These results suggest that hypermethylation might be involved in the relapse of childhood ALL.     

Aberrant DNA methylation in pediatric patients with acute lymphocytic leukemia

BACKGROUND: Aberrant methylation of promoter-associated cystosine-guanine (CpG) islands is an epigenetic modification of DNA frequently observed in adult patients with acute lymphocytic leukemia (ALL). This epigenetic modification has been associated with gene silencing, malignant transformation, and aging. It is not known whether there are epigenetic differences between pediatric patients and adult patients with ALL. METHODS: To investigate the methylation characteristics of pediatric patients with ALL and to determine whether DNA methylation can explain prognostic or biologic differences between pediatric and adult patients, the authors analyzed the methylation status of 7 promoter-associated CpG islands in 16 pediatric patients with ALL and compared them with the methylation characteristics of a cohort of adult patients with ALL. The genes analyzed included the estrogen receptor gene (ER), multidrug resistance gene 1 (MDR1), p15, C-ABL, CD10, p16, and p73. RESULTS: The mean methylation densities of ER, MDR1, CD10, p15, and C-ABL were 25.4%, 16.4%, 5.23%, 4.24%, and 4%, respectively. P16 was methylated in 11.7% of patients, and p73 was methylated in 17.6% of patients. One patient (6.2%) had methylation of 0 genes, 15 patients (93.7%) had methylation of >/= 1 gene, and 4 patients (25%) had methylation of 3-4 genes. Methylation of all these genes was < 2% (or methylation specific polymerase chain reaction negative) in nonneoplastic tissues. A significant inverse correlation was observed between methylation of CD10 and CD10 expression. No differences were observed between the methylation characteristics of pediatric patients and adult patients. CONCLUSIONS: The results indicate that DNA methylation is common in pediatric patients with ALL and that methylation of the genes studied does not account for prognostic differences between pediatric patients and adult patients with ALL.     

急性淋巴细胞白血病常见融合基因检测的研究

 【摘要】　目的　探讨联合应用多重巢式反转录聚合酶链反应（RT-PCR）技术和染色体核型分析对急性淋巴细胞白血病（ALL）常见融合基因的表达和克隆性染色体异常的检出情况。方法　采用多重巢式RT-PCR技术对189例ALL患者进行检测,同时进行染色体R或G显带。结果　189例ALL患者中69例（36.5 %）检出10种融合基因（E2A-PBX1、TEL-AML1、bcr-abl、MLL-AF4、MLL-AF6、MLL-AF9、MLL-AF10、MLL-ELL、SIL-TAL1、TLS-ERG）,染色体R或G显带可供分析的152例中,86例（56.6 %）检出染色体结构和数目异常;二者联合可使ALL克隆性染色体异常检出率增至69.3%（131例）。90例成年ALL患者中33例（36.7 %）检测阳性,其中bcr-abl 22例,未见TEL-AML1,99例儿童ALL患者中阳性36例（36.4 %）,其中TEL-AML1 24例,bcr-abl 2例。成年人组与儿童组bcr-abl 、TEL-AML1的表达率差异有统计学意义（均P＜0.01）。MLL相关融合基因、E2A-PBX1、SIL-TAL1、TLS-ERG等表达率差异无统计学意义（均P＞0.05）。66例正常核型的ALL患者检测出有bcr-abl 、TEL-AML1融合基因的存在。结论 成年人和儿童ALL融合基因表达各有侧重,多重巢式RT-PCR可用于初诊时染色体畸变的筛选,可在核型正常的ALL患者中检出隐匿的染色体易位,提供与预后相关的重要信息。     

原发胃癌中p15基因异常及启动子区甲基化状态

目的　检测胃癌组织中p15基因异常及启动子甲基化状态。方法　选择p15基因及启动子区域 ,用PCR -SSCP、MSP(甲基化特异的PCR)法和测序等方法对 10 0例胃癌患者的癌组织和癌旁组织进行检测。结果　 9%的病例具有p15基因启动子区的高甲基化 ,p15基因发现两例DNA序列改变。结论　p15基因在胃癌中起一定作用且p15基因启动子区域高甲基化是胃癌中p15基因失活的重要原因。     

Promoter hypermethylation of tumor-related genes in gastric intestinal metaplasia of patients with and without gastric cancer

Promoter hypermethylation is an alternative mechanism of gene silencing in human cancers including gastric cancer. While intestinal metaplasia (IM) is generally regarded as a precancerous lesion of the stomach, our study examines the presence of gene promoter hypermethylation in IM of patients with and without gastric cancer. We examined 31 samples of gastric cancer, 36 gastric IM (21 associated with gastric cancer and 15 from noncancer patients) and 10 normal gastric biopsies. Tissues containing foci of IM were carefully microdissected from paraffin-embedded section. Bisulfite-modified DNA was examined for gene promoter hypermethylation in DAP-kinase, E-cadherin, GSTP1, p14, p15, p16, RASSF1A and hMLH1 by methylation-specific-PCR. None of the control gastric tissues had hypermethylation detected, but gene promoter hypermethylation was frequently detected in gastric cancer and IM. The mean number of methylated genes in cancer and IM was 3.0 and 1.4, respectively (p < 0.0001). Methylation in IM from cancer patients was all associated with concurrent methylation in the corresponding tumor samples. The numbers of methylated genes were similar in IM obtained from cancer and noncancer patients. By examining the methylation patterns of these genes, 3 differential methylation patterns were recognized: hypermethylation was more frequent in cancer than in IM (DAP-kinase, p14, p15 and p16); comparable frequencies of methylation in cancer and IM (E-cadherin and hMLH1); and no methylation (GSTP1). Aberrant methylation in tumor-related genes is frequently detected in gastric IM of both cancer and noncancer patients, suggesting their early involvement in the multistep progression of gastric carcinogenesis.     

Aberrant DNA methylation of a cell cycle regulatory pathway composed of P73, P15 and P57KIP2 is a rare event in children with acute lymphocytic leukemia

Aberrant DNA methylation of multiple promoter associated CpG islands is a frequent phenomenon in acute lymphocytic leukemia (ALL). Recently, methylation of a cell cycle control pathway composed of P73, P15 and P57KIP2 has been shown to confer poor prognosis to adult patients with ALL. Using bisulfite PCR methods, we have explored the prevalence of methylation of this pathway in a cohort of children with ALL (N=20), and compared these results with those observed in a group of adult patients (N=53). P73 was methylated in 4 (20%) pediatric patients, P15 in 3 (15%), and P57KIP2 in 2 (10%). These compared to 14 (26%), p=0.5, 16 (30%), p=0.04 and 20 (37%), p=0.04, respectively in adult patients. Methylation of two or more genes was not observed in any pediatric patient, but in 15 (28%) adult patients (p=0.003). Poor survival of adult patients was associated with methylation of > or =2 genes (p=0.003). These results indicate that differences in DNA methylation of specific molecular pathways may contribute to the prognostic differences known to occur between pediatric and adult patients with ALL.