microRNA在肺癌诊断、治疗及预后判断中的作用

肺癌是全世界因肿瘤导致死亡的最主要原因。尽管肺癌发生的分子网络在蛋白和基因水平上已经部分得到阐明,但是其高死亡率仍没有明显改善。微小RNAs(miRNAs)是一大类小的内源性非编码RNAs,通过与靶mRNA3’端非翻译区部分互补配对,在翻译后水平调节靶基因的表达。miRNAs在肺脏的发育过程中发挥多种作用,其异常表达可能导致肺癌的发生。肺癌中miRNAs的异常表达模式,以及miRNAs在血清中的稳定存在,都使miRNAs有望成为新型的临床生物标记物用于肺癌的诊断和预后。miRNAs还可以直接作为原癌基因或抑癌基因在肺癌发生和发展过程中起重要作用,调节具有致癌或抑癌功能的miRNAs可能成为肺癌治疗的新方法。该文就miRNAs在肺癌诊断、预后和治疗中的作用进行综述。     

基于悬浮点阵技术的新型EGFR基因突变检测方法的建立和应用

目的构建基于悬浮点阵技术的新型表皮生长因子受体(EGFR)基因突变的检测方法,检测并分析非小细胞肺癌(NSCLC)组织中EGFR酪氨酸激酶抑制剂(TKI)相关的EGFR基因突变状况。方法选取61例经影像学和病理学检查确诊的NSCLC患者作为研究对象,手术后留取新鲜肺癌组织,提取基因组DNA,采用多重聚合酶链式反应(PCR)对组织样本的EGFR基因18、19、20和21号外显子中包含8种高频突变位点的特异片段进行扩增,并经多重连接酶检测反应(LDR)以特异性探针对扩增片段中的突变进行连接,再基于悬浮点阵技术平台对连接产物进行检测,最后通过DNA测序对检测结果进行验证。结果在61例NSCLC组织样本中,运用PCR-LDR联合悬浮点阵技术共检出EGFR基因突变19例,突变率为31.1%,其中外显子19缺失突变E746-A750 del(1)、E746-A750 del(2)各1例,外显子20点突变T790M 1例,外显子21点突变L858R 14例、L861Q2例,经DNA测序验证完全正确。结论运用PCR-LDR联合悬浮点阵技术初步建立了一种新型的EGFR基因突变检测方法,能同时准确检测EGFR基因中的8种高频突变...     

新型高通量肺癌相关基因甲基化检测方法的建立

Objective To explore a new high-throughput method with internal standards for analyzing the methylation profiles of lung cancer related genes. Methods The promoter sequences of 7 lung cancer related genes were cloned into plasmids and the target segments were amplified by their special primers respectively. The products were treated with M. Sss Ⅰ methylase and bisulfite. The multiplex ligation PCR method was established by designing probes containing CpGpCpG(for methylatedsequence) at the 3' ends and choosing the optimal ligation enzyme, annealing and ligation temperatures. The standard calibrators and clinic samples were tested by fluid chip platform. The results were validated by methylationspecific PCR. Results We successfully set up the standard calibrators for methylation and unmethylaiton of 7 lung cancer related genes and established a multiplex ligation PCR combined with fluid chip method, which was used to detect methylation status of 7 genes simultaneously. The fluorescence value of p16INK4A, APC,DAPK, RARIβ, RASSF1 A, MGMT and GSTP1 methylation standard calibrators were 863,909,703,701,901,1 060 and 885, much higher than that of unmethylation standard calibrators. The results were consistent with the results of methylation-specific PCP. ConclusionThe new high-throughput method can be used to evaluate the methylation status of 7 lung cancer related genes simultaneously and might be useful for clinical practice.     

新型高通量肺癌相关基因甲基化检测方法的建立

Objective To explore a new high-throughput method with internal standards for analyzing the methylation profiles of lung cancer related genes. Methods The promoter sequences of 7 lung cancer related genes were cloned into plasmids and the target segments were amplified by their special primers respectively. The products were treated with M. Sss Ⅰ methylase and bisulfite. The multiplex ligation PCR method was established by designing probes containing CpGpCpG(for methylatedsequence) at the 3' ends and choosing the optimal ligation enzyme, annealing and ligation temperatures. The standard calibrators and clinic samples were tested by fluid chip platform. The results were validated by methylationspecific PCR. Results We successfully set up the standard calibrators for methylation and unmethylaiton of 7 lung cancer related genes and established a multiplex ligation PCR combined with fluid chip method, which was used to detect methylation status of 7 genes simultaneously. The fluorescence value of p16INK4A, APC,DAPK, RARIβ, RASSF1 A, MGMT and GSTP1 methylation standard calibrators were 863,909,703,701,901,1 060 and 885, much higher than that of unmethylation standard calibrators. The results were consistent with the results of methylation-specific PCP. ConclusionThe new high-throughput method can be used to evaluate the methylation status of 7 lung cancer related genes simultaneously and might be useful for clinical practice.     

流式荧光法检测NSE的应用评价

建立流式荧光法定量检测神经元特异性烯醇化酶(NSE)的正常参考值,评估其在肺癌诊断中的应用价值。采用流式荧光法检测176例临床血清样本的NSE水平,并与罗氏Elecsys电化学发光法检测结果进行比较,确定两种方法之间的可比性。进一步采用流式荧光法检测668例健康者血清NSE水平,建立健康人群的NSE正常参考值。最后,以流式荧光法检测242例肺癌患者、108例肺部良性疾病者和147例健康者血清NSE水平,评价流式荧光法检测NSE的应用。结果显示,176例临床样本的检测结果表明流式荧光法和电化学发光法相关性良好(Y=0.9746X-0.2075,R2=0.9653),二者具有可比性。根据668例健康者检测结果的第95%百分位数(17.07ng/ml)确定流式荧光法的NSE正常参考值,与罗氏Elec-sys 2010电化学发光法的相似。以NSE>17.07ng/ml判断为阳性,NSE在小细胞肺癌患者中的阳性率显著高于肺鳞癌、肺腺癌患者(P<0.01)。建立了流式荧光法检测NSE的正常参考值,并初步评估了流式荧光法检测NSE在肺癌诊断中的应用,为流式荧光法检测NSE的合理应用和广泛推广奠定了基础。     

甲巯咪唑与甲氨蝶呤对Graves病免疫功能的影响

以健康人群为对照，Graves病患者分成甲巯咪唑(MMI)组(MMI 20mg/d)和甲氨蝶呤(MTx)组(MMI20mg/d MTX7．5mg/w)，测得患者组CD4、Th增高而Tc低下，治疗6周后该异常虽无明显改变，可溶性白细胞介素2(sIL-2)受体和甲状腺自身抗体下降，但组间差异无显著性。提示Graves病治疗前存在免疫异常，MMI及其加用MTX治疗6周使sIL-2受体及甲状腺自身抗体明显下降，加用MTX未见对免疫功能的影响优于单用MMI。     

结核分枝杆菌保护性肽表位KLIANNTRV多聚体的免疫原性研究

抗结核病的保护性免疫机制目前仍不清楚，细胞免疫尤其是循环及感染部位的T淋巴细胞应答被认为起着重要作用。我们采用分子生物学的方法重组表达了结核分枝杆菌保护性肽表位KLIANNTRV二聚体、四聚体和八聚体，并采用结核菌素纯蛋白衍生物（PPD）试验阳性健康个体的外周血单个核细胞验证其免疫原性，为设计新型结核疫苗奠定良好的基础。     

细胞外基质蛋白1在肿瘤中的表达及意义

目的:探讨细胞外基质蛋白1(extracellular matrix protein 1,ECM1)在肿瘤组织中的表达及意义.方法:应用免疫组织化学EnVision法,检测肝脏、乳腺、直肠组织(正常组织和肿瘤组织)中ECM1的表达,比较正常组织和肿瘤组织淋巴结转移肿瘤和淋巴结未转移肿瘤之间的ECM1表达差异.结果:ECM1的表达如下:正常肝组织阳性率20.0%(4/20),肝癌组织阳性率85.4%(70/82);正常乳腺组织阳性率9.1%(1/11),乳腺癌组织阳性率60.0%(18/30),其中淋巴结(-)者为37.5%(6/16),淋巴结( )者为85.7%(12/14);正常直肠组织阳性率11.8%(2/17),直肠癌组织阳性率54.5%(18/33),其中淋巴结(-)者为33.3%(6/18),淋巴结( )者为80.0%(12/15).统计分析表明,对ECM1的表达,肿瘤组织高于正常组织,淋巴结转移性肿瘤高于非转移性(P＜0.01).结论:ECM1在肿瘤组织中过表达,并且与肿瘤的转移相关.     

肺癌分子诊断学研究进展

肺癌的分子诊断作为影像学和细胞学筛查策略的重要补充,其内容不仅包括早期诊断,还包括预后指标及靶向治疗的预测等。该文拟对当前肺癌分子诊断的研究热点基因突变与靶向治疗预测、miRNA、肿瘤干细胞、甲基化及相关检测方法进行综述。