自体积液癌细胞培养及药敏试验对晚期肺腺癌化疗的临床应用价值

背景与目的晚期肺癌的化疗效果存在极大的个体差异,如何选择最佳化疗方案,实现肺癌化疗的个体化、效果最大化是值得探索的课题。本研究旨在检验自体积液中进行癌细胞培养及药敏试验对于合并胸腔或心包积液的肺腺癌患者优化化疗方案的临床应用价值。方法收集50例并发恶性胸腔和/或心包积液的肺腺癌初治患者,经闭式引流控制积液。其中25例（药敏组）于无菌条件下留取积液300 mL-500 mL,肿瘤细胞通过自体积液（xeno-free）平均11天的细胞培养而获得,继而针对8种临床常用化疗药物行药敏试验,通过CCK-8进行敏感性检测,根据试验结果选择最优化疗方案进行全身化疗;另25例（对照组）进行经验性化疗。结果4个周期化疗后,药敏组部分缓解（partial response, PR）17例（68.0%）、稳定（stable disease, SD）5例（20.0%）,客观缓解率（objective response rate, ORR）68.0%,疾病控制率（disease control rate, DCR）88.0%;对照组PR 9例（36.0%）、SD 7例（28.0%）,ORR为36.0%,DCR为64.0%。比较两组ORR和DCR,差异均有统计学意义（P<0.05）。至随访截止,药敏组死亡21例,对照组死亡22例。药敏组无进展生存期（progression-free survival, PFS）平均10.0个月,总生存期（overall survival, OS）平均30.2个月;对照组PFS平均5.8个月,OS平均21.2个月。比较两组PFS和OS,差异均有统计学意义（P<0.05）。两组毒副反应轻微、可控。结论自体积液癌细胞培养及药敏试验有利于晚期肺腺癌患者化疗方案的合理选择。     

血管紧张素转化酶(ACE)试剂盒的性能评价

目的自建血管紧张素转化酶检测系统的性能评价。方法依据临床和实验室标准化协会(CLSI)标准中EP5-A2,EP9-A文件的要求,采用全自动生化分析仪对九强公司的ACE试剂盒的精密度、线性、与比对试剂的相关性以及可报告范围等一些性能进行评价。结果自建系统血清ACE高低值的批内CV分别为6.87%和2.39%,批间CV分别为6.09%和1.81%,日间CV分别为8.00%和2.8%均在厂家提供的小于10%的范围内;线性结果R2=0.99;与比对试剂相关系数r=0.990 56,且平均偏倚为8.55%有良好的相关性;血清ACE检测试剂盒可报告范围为9.0U/L-600U/L。结论自建ACE检测系统各项性能可以满足临床应用的要求。     

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.     

腺苷脱氨酶和癌胚抗原联合检测在结核性与恶性肿瘤胸腔积液鉴别诊断上的价值

目的为鉴别结核性和恶性肿瘤胸腔积液提供一种比较快速及有实用价值的检测方法。方法采用速率法和化学发光法分别检测65例结核性胸腔积液患者和60例恶性肿瘤胸腔积液患者胸腔积液的腺苷脱氨酶(ADA)活性和癌胚抗原(CEA)含量。结果结核组及恶性肿瘤组胸腔积液ADA活性分别为(52.9±22.8)U/L和(13.8±5.7)U/L,二者比较差异有统计学意义(P0.01)。恶性肿瘤组胸腔积液CEA含量为(37.8±17.6)ng/mL,明显高于结核组[(7.9±2.8)ng/mL](P0.01)。结论胸腔积液ADA与CEA对结核性和恶性肿瘤胸腔积液具有鉴别诊断价值。     

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。     

新型高通量肺癌相关基因甲基化检测方法的建立

目的 建立一种包含甲基化标准品的新型高通量肺癌相关基因的甲基化检测系统.方法 2009年3-6月上海交通大学医学院附属胸科医院经病理学和影像学确认的非小细胞肺癌患者8例,其中男7例,女1例,平均年龄(57±11)岁,手术后留取肺癌组织及外周血.男性健康志愿者1名,年龄35岁,抽取外周血20 ml,用以分离单个核细胞提取DNA制备质粒.对7个肺癌相关基因的启动区进行克隆,用各基因的特异性引物对克隆到质粒中的各基因片段再次进行扩增,PCR产物用甲基化酶M.sss Ⅰ和亚硫酸盐处理.通过设计3'末端CpGpCpG探针来识别甲基化模板,并经最佳连接酶、最佳退火温度及连接温度的选择,建立一套多重连接PCR体系,最后运用液相芯片平台对扩增的标准品和临床标本进行检测.利用甲基化特异性PCR检测新系统的可靠性.结果 成功的构建了7个基因的甲基化和非甲基化标准品,并建立了多重PCR偶联液相芯片的快速检测系统,p16INK4A、APC、DAPK、RARβ、RASSF1 A、MGMT和GSTP1甲基化标准品的荧光信号值分别为863、909、703、701、901、1 060和885,与非甲基化标准品区分明显.抽提8例患者的肺癌组织DNA,对其中检测出阳性频率较高的基因进行甲基化特异性PCR,产物电泳结果 与新建甲基化检测系统检测结果 完全一致.结论 本研究建立的新型检测系统能同时准确地检测7个基因甲基化状态,有望应用于临床标本的检测.     

10个肺癌相关基因多重甲基化特异性PCR检测方法的建立

目的建立10个肺癌相关基因的多重甲基化特异性聚合酶链反应(PCR)检测方法并运用于临床标本的检测。方法选取19例肺癌患者病灶组织标本,患者中男性16例,女性3例;平均年龄57.3岁。1例肺部良性病变患者组织标本为男性,年龄52岁。通过质粒构建及甲基化转移酶和亚硫酸盐修饰制备10个基因的甲基化和非甲基化标准品。通过选择甲基化特异性引物来识别甲基化模板,挑选10对甲基化特异性引物并平均分成2组,经最佳聚合酶及最佳退火温度的选择,建立多重甲基化特异性PCR体系,并应用于19例肺癌组织的检测,计算10个基因的甲基化率。结果在成功构建10个基因的甲基化和非甲基化标准品的基础上,建立了它们的多重甲基化特异性PCR检测方法。临床标本检测结果显示10个肺癌相关基因的甲基化率分别为p16INK4A 92%,H-cadherin 89%,E-cadherin和DAPK 76%,TIMP-3 63%,RARβ50%,MGMT 39%,RASSF1A 21%,GSTP1 11%,hMLH1 0%。2次实验结果重复性较好。结论这种分2组多重甲基化特异性PCR的方法能一次性检测10个基因甲基化状态,可以应用于临床标本的检测。