先天性心脏病相关GATA5基因突变研究

目的:识别先天性心脏病(CHD)相关GATA5基因新突变。方法:收集100例无血缘关系的CHD患者和200名无血缘关系且种族匹配的健康对照者的临床资料和血标本。抽提基因组DNA,通过聚合酶链反应扩增GATA5基因的编码外显子及其剪接位点,采用双脱氧核苷链末端合成终止法对全部扩增片段进行测序。将所测的序列与GenBank数据库中的GATA5基因序列进行比对以发现GATA5基因突变。应用多序列比对软件ClustalW评估突变氨基酸的保守性,应用致病性预测软件MutationTaster预测突变的致病性。结果:在2例CHD患者各发现1个新的GATA5基因杂合错义突变,突变率为2%。其中1个是GATA5基因编码核苷酸序列第395位的鸟嘌呤(guanine,G)变为胸腺嘧啶(thymine,T),即c.395G>T突变;另一个是GATA5基因编码核苷酸序列第991位的胞嘧啶(cytosine,C)变为腺嘌呤(adenine,A),即c.991C>A突变。多序列比对显示2种突变氨基酸在进化上均高度保守。致病性预测显示2种突变均有致病性。结论:本研究发现了CHD相关GATA5基因新突变,有助于揭示CHD新的分子机制。     

DNA甲基化与肿瘤

肿瘤发生、发展的分子生物学本质是细胞内遗传调控和表观遗传调控(epigenetic regulation)的紊乱。国内外对基因组所携带的遗传信息与疾病的关系已有深入的研究。相对而言,染色质所携带的表观遗传信息在疾病发生、发展中的重要作用才刚刚开始被认识。影响基因转录活性而不涉及DNA序列改变的基因表达调控方式称为表观遗传调控,其分子基础是DNA甲基化以及染色质的化学修饰和物理重塑。大量的临床和基础研究结果表明环境因素在肿瘤发生、发展中有巨大的影响,而表观遗传调控在遗传因素和环境因素的互动关系中起着桥梁的作用。     

特发性房颤相关GATA5基因新突变的识别

目的:识别特发性房颤(AF)相关GATA5基因新突变. 方法:收集120例无血缘关系的特发性AF患者和200名无血缘关系且种族匹配的健康对照者的临床资料和血标本.抽提基因组DNA,通过聚合酶链反应扩增AF候选基因GATA5的全部外显子及其两侧的部分内含子,采用双脱氧核苷链末端合成终止法对全部扩增片段进行测序.将所测的序列与GenBank数据库中的GATA5基因序列进行比对,以发现GATA5基因突变.应用多序列比对软件ClustalW评估突变氨基酸的保守性,应用致病性预测软件MutationTaster预测突变的致病性. 结果:在2例AF患者各发现1个新的GATA5基因杂合错义突变,突变率约为1.67％.突变分别为GATA5基因编码核苷酸序列第668位的腺嘌呤(adenine,A)变为胸腺嘧啶(thymine,T),即c.668A＞T突变;第863位的A变为胞嘧啶(cytosine,C),即c.863A＞C突变.多序列比对显示2种突变氨基酸在进化上均高度保守,致病性预测表明2种突变均有致病性. 结论:本研究识别出AF相关GATA5基因新突变,有助于揭示AF发生的分子机制.     

转录因子GATA—2基因在ANLL、CML和MDS中的表达

目的：了解GATA－2基因在急性非淋巴细胞白血病（ANLL）、慢性粒细胞白血病（CML）和骨髓增生异常综合征（MDS）中的表达情况。方法：利用RT－PCR扩增ANLL、CML和MDS等246例患者外周血单个核细胞中GATA－2基因。结果：GATA－2基因在ANLL、CML和MDS中的表达率分别为88．4％、81．6％和97％,差异与正常人有高度显著性（P＜0．01）,结论：绝大多数髓系白血病和MDS标本表达GATA－2基因,GATA－2可能是白血病细胞的增殖过程中所需要的转录因子之一。     

SLE患者PBMCs中T-bet、GATA3和Foxp3 mRNA的表达变化及意义

目的检测系统性统斑狼疮（SLE）患者单个核细胞（PBMCs）中转录因子T-bet、GATA3和Foxp3表达情况,并探讨其意义。方法采集系统性红斑狼疮（SLE）患者外周血单个核细胞（PBMCs）,提取总RNA,利用SYBRGreenⅠ逆转录荧光定量PCR方法,以β-actin为内对照基因,检测PBMCs中T-bet、GATA3和Foxp3mRNA的相对表达水平。结果SLE患者PBMCs中T-bet、GATA3、Foxp3mRNA表达水平低于正常对照组,且活动期高于稳定期。结论SLE患者PBMCs中转录因子T-bet、GATA3和Foxp3表达降低;三者可能参与SLE的发病机制。     

先天性房间隔缺损相关GATA6基因新突变的识别

目的：识别先天性房间隔缺损（ASD）相关GATA6基因新突变。方法：收集110例先天性AsD患者和200名健康对照者的临床资料和血标本,使用DNA纯化试剂盒提取基因组DNA。通过聚合酶链反应扩增GATA6基因的编码外显子和其两侧的部分内含子,应用双脱氧核苷链末端合成终止法进行DNA测序。将所测序列与GenBank数据库中的GATA6基因序列进行比对,识别GATA6基因突变。分别借助在线程序MUSCLE和MutationTaster评估突变氨基酸的保守性和致病性。结果：在1例家族史阴性的先天性ASD患者发现了1种新的GATA6基因杂合错义突变,即P．Q363E突变,突变率约为0．91％。该突变不存在于200名健康对照者,多物种序列比对显示,被改变氨基酸在进化上高度保守,致病性预测表明这种变异为致病性突变。结论：发现ASD相关GATA6基因新突变,有助于揭示ASD新的分子机制。     

肝细胞癌患者PBMCs中T-bet、GATA3和Foxp3 mRNA水平及其意义

目的探讨肝细胞癌患者外周血单个核细胞(PBMC)中T-bet、GATA3和Foxp3 mRNA水平变化及意义。方法在肝细胞癌患者20例和健康对照人群10例,采用RT-PCR法检测PBMC中T-bet、GATA3和FoxP3mRNA水平。结果 HCC患者和健康对照T-bet水平分别为0.554±0.030和0.514±0.071(P=0.391);HCC患者和健康对照GATA3水平分别为0.956±0.030和0.535±0.028(P<0.01);HCC患者和健康对照FoxP3水平分别为0.976±0.073和0.772±0.083(P<0.01);HCC患者和健康对照T-bet/GATA3比值分别为0.697±0.078和0.963±0.133(P<0.01)。结论肝细胞癌患者PBMC中GATA3和FoxP3 mRNA水平上调,可能参与了HCC的发生和发展过程。     

中国儿童急性淋巴细胞白血病GATA3基因单核苷酸多态性与预后的相关性研究

目的 研究中国儿童急性淋巴细胞白血病(ALL)患儿GATA3基因单核苷酸多态性(SNP)分布特点及其对预后的影响.方法 利用TaqMan探针荧光定量PCR法检测上海儿童医学中心2009-05～2014-12收治的678例ALL患者GATA3基因rs3781093和rs3824662两个SNP位点,分析其与早期治疗反应、...     

替米沙坦对压力超负荷大鼠心肌内皮素-1表达和GATA DNA结合活性的影响

目的 研究压力超负荷心肌肥厚大鼠心肌组织(GATA)DNA结合活性和内皮素(ET)-1蛋白表达的变化及其意义,观察替米沙坦对压力超负荷心肌肥厚大鼠血流动力学参数[收缩压(SBP)、舒张压(DBP)和平均压(MBP)]、左室质量指数(LVMI)、心肌细胞横径(DC)、心肌组织GATA DNA结合活性和ET-1蛋白表达的影响.方法 腹主动脉缩窄法建立压力超负荷大鼠心肌肥厚模型.健康SD大鼠24只,随机分为三组:假手术组(n=8);手术组(n=8);替米沙坦组(手术后第7天给替米沙坦3 mg·kg-1·d-1灌胃4 w,n=8).测定大鼠SBP、DBP、MBP、LVMI及DC;免疫组化半定量检测心肌组织ET-1蛋白的表达,电泳迁移率变动分析检测ET-1与GATA的特异性结合及GATA DNA结合活性的变化.结果 ①与假手术组比较,手术组SBP、DBP、MBP、LVMI和DC均显著升高(P均<0.05);光镜下见心肌实质、间质结构改变;心肌组织ET-1蛋白表达和GATA DNA结合活性均明显增加(P均<0.05);心肌组织ET-1蛋白表达与LVMI和DC均呈显著正相关(r=0.895,r=0.899,P均<0.01);②与手术组比较,替米沙坦组SBP、MBP、LVMI、DC、心肌组织ET-1蛋白表达和GATA DNA结合活性均明显降低(P均<0.05).结论 (1)GATA DNA结合活性增加使ET-1蛋白表达增加与压力超负荷大鼠的心肌肥厚有关;(2)替米沙坦能够抑制压力超负荷大鼠的心肌肥厚,其机制可能与替米沙坦降低GATA DNA结合活性从而下调ET-1蛋白表达有关.     

JAM3基因甲基化是食管鳞状细胞癌潜在的分子标志物

目的研究JAM3基因启动子区在食管癌中的甲基化情况及其表达调控机制,探讨JAM3基因启动子区异常甲基化作为食管鳞状细胞癌的潜在诊断标志物和治疗靶标。方法应用半定量RT-PCR、甲基化特异性PCR等技术对7个食管癌细胞系(KYSE140、KYSE150、KYSE410、KYSE450、COLO680N、KYSE520和TE13)、5例正常食管黏膜组织和83例原发性食管鳞状细胞癌组织进行分析。结果JAM3 mRNA在KYSE520、KYSE140、KYSE450细胞中高表达,这些细胞的JAM3基因启动子区呈非甲基化状态。JAM3 mRNA在KYSE410、COLO680N、TE13、KYSE150细胞中表达缺失,且其基因启动子区在这些细胞中呈完全甲基化。经过5-Aza-dc处理后,JAM3基因在KYSE410、COLO680N、TE13、KYSE150细胞中恢复表达。这些结果表明,JAM3在食管癌细胞中的表达受启动子区甲基化的调控。JAM3基因启动子区在5例正常食管黏膜组织中呈非甲基化状态(0/5),而在原发性食管鳞状细胞癌中其甲基化率为50.6%(42/83),且JAM3甲基化与肿瘤的位置相关(P<0.05)。结论JAM3在食管鳞状细胞癌中频繁发生甲基化,JAM3基因的表达受启动子区甲基化的调控,JAM3基因是潜在的食管癌诊断标志物和治疗靶标。     

胃癌HOXB6基因异常甲基化及其表达

目的 检测胃癌组织中HOXB6基因是否存在异常甲基化，并探讨其与基因表达及临床因素的关系。方法 用联合重硫酸盐限制性分析法（COBRA法）对7种胃癌细胞系和73例胃癌中层得的胃镜下活检组织进行HOXB6基因的甲基化定量检测，并采用逆转录聚合酶链反应方法检测基因的表达及脱甲基化处理后基因的再表达，结果 7种胃癌细胞系中有4种异常甲基化，异常甲基化的细胞系基因表达减少或消失，并且脱甲基化处理后基因可以重新表达，73例胃癌患者的癌部位和非癌部位的活检组织中，20例癌组织甲基化阳性（27.40%)，而非癌组织中无1例阳性。结论 胃癌中HOXB6基因的异常甲基化与基因表达抑制密切相关。并且脱甲基化后基因可以再表达；胃癌组织中HOXB6基因异常甲基化具有高度特异性，可作为肿瘤标志物用于临床诊断。     

Combined detection of plasma GATA5 and SFRP2 methylation is a valid noninvasive biomarker for colorectal cancer and adenomas

AIM:To investigate GATA5,SFRP2,and ITGA4 methylation in plasma DNA as noninvasive biomarkers for colorectal cancer(CRC) or adenomas.METHODS:There were 57 CRC patients,30 adenomas patients,and 47 control patients enrolled in this study.Methylation-specific polymerase chain reaction was used to determine the promoter methylation status of GATA5,SFRP2,and ITGA4 genes in plasma DNA,and their association with clinical outcome in CRC.The predictive ability of GATA5,SFRP2,and ITGA4 methylation,individually or in combination,to detect CRC or adenomas was further analyzed.RESULTS:Hypermethylated GATA5 was detected in plasma in 61.4%(35/57) of CRC cases,43.33%(13/30) of adenoma cases,and 21.28%(10/47) of control cases.The hypermethylation of SFRP2 was detected in 54.39%(31/57),40.00%(12/30),and 27.66%(13/47) in plasma samples from CRC,adenomas,and controls,respectively.ITGA4 methylation was detected in 36.84%(21/57) of plasma samples of CRC patients and in 30.00%(9/30) of plasma samples from patients with colorectal adenomas,and the specificity of this individual biomarker was 80.85%(9/47).Moreover,GATA5 methylation in the plasma was significantly correlated with larger tumor size(P =0.019),differentiation status(P =0.038),TNM stage(P =0.008),and lymph node metastasis(P =0.008).SFRP2 and ITGA4 methylation in plasma significantly correlated with differentiation status(SFRP2,P =0.012; ITGA4,P =0.007),TNM stage(SFRP2,P =0.034; ITGA4,P =0.021),and lymph node metastasis(SFRP2,P =0.034; ITGA4,P =0.021).From the perspective of predictive power and cost-performance,using GATA5 and SFRP2 together as methylation markers seemed the most favorable predictor for CRC(OR =8.06;95%CI:2.54-25.5; P < 0.01) and adenomas(OR =3.35; 95%CI:1.29-8.71; P =0.012).CONCLUSION:A combination of GATA5 and SFRP2 methylation could be promising as a marker for the detection and diagnosis of CRC and adenomas.     

Hypermethylation and aberrant expression of Wnt antagonist secreted frizzled-related protein 1 in gastric cancer

AIM： To identify the methylation of secreted frizzled-related protein 1 （SFRP1） in gastric cancer and to investigate the aberrant expression of SFRP1 and its correlation with the clinical pathological features of patients.
METHODS： We determined SFRP1 methylation and SFRP1 mRNA expression in 3 gastric cancer cell lines SGC-7901, BGC-823, HGC-27, from 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens by methylation-specific （MSP） PCR and RT-PCR respectively. Fisher＇s exact test was used to analyze the statistical association between clinical pathological data and aberrant expression of SFRP1.
RESULTS： In 3 cancer cell lines, BGC-823 and HGC-27 had methylated SFRP1 and lost SFRP1 mRNA expression. After treatment of BGC-823 and HGC-27 with the demethylating agent, 5-aza-2′-deoxycytidine, SFRP1 was re-expressed. In 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens, hypermethylation of SFRP1 was detected in 23 （44%） and 8 （15%） specimens respectively （x^2= 10.34, P 〈 0.01）. Loss of SFRP1 expression was detected in 17（33%） and 6 （12%） specimens respectively （x^2= 6.75, P 〈 0.01）. There was a significant correlation between SFRP1 hypermethylation and SFRP1 expression loss. SFRP1 expression was also correlated significantly with tumor stage and lymph node status, but not with patient sex, age and histological type.
CONCLUSION： SFRP1 inactivation is a common and early event caused mainly by hypermethylation in gastric cancer. SFRP1 expression loss may be correlated with tumor metastasis in primary gastric cancer.     

结肠癌中骨形成蛋白-6启动子异常甲基化

目的研究骨形成蛋白-6（BMP-6）启动子在结肠癌细胞中的甲基化状态。方法运用甲基化特异PCR（MSP）法检测30例原发结肠癌和癌旁组织中BMP-6的甲基化水平。甲基化修饰后亚硫酸盐测序PCR（BSP）分析结肠癌细胞COLO-205细胞BMP一6启动子CpG岛甲基化状态。提取细胞RNA,通过实时定量PCR（real time-PCR）检测BMP-6mRNA在不同浓度的DNA甲基化酶抑制剂5＇-杂氮-2’-脱氧胞嘧啶（5-aza-dC）处理COLO-205细胞后BMP-6mRNA的改变情况。结果原发性结肠癌的甲基化异常检出率是33．3％（10／30）。BMP-6在COLO-205细胞中存在高甲基化状念,COLO-205细胞经5-aza-dC处理后BMP-6mRNA表达明显上调。结论BMP-6在原发性结肠癌中存在甲基化异常,说明BMP-6鹾闪甲基化异常可能参与结肠癌的发生。     

Transcriptional silencing of Dickkopf gene family by CpG island hypermethylation in human gastrointestinal cancer

AIM： To clarify alterations of Dickkopfs （Dkks） and Kremen2 （Krm2） in gastrointestinal cancer.
METHODS： We investigated the expression profiles and epigenetic alterations of Dkks and Krm2 genes in gastrointestinal cancer using RT-PCR, tissue microarray analysis, and methylation specific PCR （MSP）. Cancer cells were treated with the demethylating agent and/or histone deacetylase inhibitor. WST-8 assays and/n y/tro invasion assays after treatment with specific siRNA for those genes were performed.
RESULTS： Dkks and Krm2 expression levels were reduced in a certain subset of the gastrointestinal cancer cell lines and cancer tissues. This was correlated with promoter hypermethylation. There were significant correlations between Dkks over-expression levels and beta-catenin over-expression in colorectal cancer. In colorectal cancers with beta-catenin over-expression, Dkk-1 expression levels were significantly lower in those with lymph node metastases than in those without. Down-regulation of Dkks expression by siRNA resulted in a significant increase in cancer cell growth and invasiveness in vitro.
CONCLUSION： Down-regulation of the Dkks associated to promoter hypermethylation appears to be frequently involved in gastrointestinal tumorigenesis.     

内镜下黏膜切除术在治疗早期食管癌和重度不典型增生中的应用

目的探讨内镜下黏膜切除术（EMR）治疗早期食管癌、重度不典型增生的应用价值。方法对我院2004年2月～2009年4月经色素内镜筛查且活检证实为早期食管癌及重度不典型增生的32例患者,在静脉麻醉下进行内镜下黏膜切除术透明帽法治疗,其中早期癌8例,重度不典型增生24例。结果对早期食管癌及重度不典型增生的32例患者进行内镜下黏膜切除术透明帽法治疗,成功27例,小量出血2例,无穿孔及狭窄等严重并发症。结论严格筛选患者行内镜下黏膜切除术透明帽法治疗早期食管癌、癌前病变是安全而有效的方法。     

抑癌基因启动子过甲基化对肺癌临床研究的意义

抑癌基因启动子过甲基化是除基因突变和染色体物质缺失外抑癌基因失活的第3种机制,某些情况下是抑癌基因失活的惟一机制.目前抑癌基因启动子过甲基化已被用于肺癌的诊断、筛查和监测肿瘤复发的研究.本文对抑癌基因启动子过甲基化导致肺癌发生的基础、研究过甲基化的方法、过甲基化研究在临床的应用、逆转过甲基化的探讨及目前研究的局限性作一综述.     

Hypermethylation of TGF-β1 gene promoter in gastric cancer

AIM:To examine transforming growth factor-β1(TGF-β1)promoter methylation in gastric cancer and to determine if Helicobacter pylori(H.pylori)or interleukin(IL)-1β could induce TGF-β1 hypermethylation in vitro.METHODS:We examined the frequency and extent of TGF-β1 promoter methylation using methylationspecific PCR in the gastric tissues from 47 gastric cancer patients and 39 non-gastric cancer subjects.H.pylori infection was confirmed by a positive result from either a serological test,histological analysis or C13urea breath test.GES-1 and MKN-45 cells co-cultured with H.pylori or treated with IL-1β for 12,24 and 48 h in vitro tested the effects of H.pylori or IL-1β on TGF-1β.RESULTS:Twenty-four/forty-seven(51%)cases of gastric cancer(GC)tissues showed TGF-β1 promoter methylation,15/47(31.9%)cases of matched noncancerous gastric mucosa tissues from the GC patients,and 11/39(28%)case of the normal gastric mucosa tissues from non-GC subjects showed TGF-β1 promoter methylation(51%vs 28%,P<0.05).Significantly higher levels of methylation of TGF-β1 were found in the tumor tissues than in non-tumor tissues from GC patients(0.24±0.06 vs 0.17±0.04,P<0.05)and normal gastric tissues from non-GC subjects(0.24±0.06 vs 0.15±0.03,P<0.05).TGF-β1 methylation was found in 48.3% of H.pylori-positive gastric mucosal tissues whereas only 23.1% of H.pylori-negative gastric mucosal tissues showed TGF-β1 methylation(48.3%vs 23.1%,P<0.05).IL-1β appeared to induce a dose-dependent methylation of TGF-β1 and the strongest methylation was observed in GES-1 cells treated with 2.5 ng/mL of IL-1β for 48 h.Further studies showed that pre-treatment of GES-1 cells with 20ng/mL IL-1RA for 1 h could partially abolish the effect of IL-1β on TGF-β1 methylation.Infection of GES-1cells by H.pylori was not found to induce significant TGF-β1 promoter methylation.CONCLUSION:Our data revealed that TGF-1 promoter is methylated in GC patients.IL-1β may be an important mediator for H.pylori induced gene methylation during GC