肿瘤型M2-PK、APC、K-ras检测在结直肠癌诊断中的意义

目的检测结直肠癌患者粪便和血清的M2型丙酮酸激酶(M2-PK)、APC、K-ras水平,并分析M2-PK、APC、K-ras的水平与结直肠癌病理T分期的关系。方法将2015年1-10月在该院手术治疗的结直肠癌患者200例纳入该研究作为患者组,另外选取健康体检者100例作为对照组。采用酶联免疫吸附测定(ELISA)检测结直肠癌患者血清和粪便的M2-PK、APC、Ras水平,并结合患者的病理资料对M2-PK、APC、K-ras的水平与结直肠癌病理T分期的关系进行分析。结果结直肠癌患者血清和粪便的M2-PK、APC、K-ras的表达水平分别高于对照组(P0.05)。M2-PK、APC、K-ras的表达与结直肠癌的病理T分期有密切关系,且临床病理T分期较晚的患者M2-PK、APC、K-ras的表达水平更高(P0.05)。结论结直肠癌患者血清和粪便的M2-PK、APC、K-ras水平较高且与肿瘤的临床病理T分期有关。M2-PK、APC、K-ras可作为早期诊断结直肠癌的分子标志物。     

结直肠癌诊断指标的研究进展

结直肠癌在我国发病日渐增多,结肠镜检查尽管是诊断结直肠癌的金标准,但为侵入性检查,易造成患者的痛苦和不适,且可能引起出血、穿孔等并发症,不易被人们接受,而检测血液和粪便标志物的非侵入筛查更易被人们认可。目前粪便隐血和血清糖类抗原(CA)检测是最常用的筛查方法,近年来的研究显示肿瘤M2型丙酮酸激酶(tM2-PK)在结直肠癌筛查中有一定的应用价值,随着分子生物学研究的进展,表观遗传学中启动子区的高甲基化和微小RNA(miRNA)的异常表达在血液和粪便中的检测有望提高结直肠癌的早期发现率,但仍需大量的临床研究予以证实。     

tM2-PK在结直肠癌的应用价值临床研究

目的研究肿瘤型丙酮酸激酶(tM2-PK)在结直肠癌患者表达情况及其与结直肠癌临床病理特征的关系。方法以49例结直肠癌患者为研究对象,免疫组化检测癌组织以及癌旁组织tM2-PK表达情况,并研究癌组织tM2-PK与患者血清tM2-PK水平相关性,分析tM2-PK与结直肠癌患者病理特征关系。结果 49例结直肠癌患者癌组织与癌旁组织tM2-PK阳性率分别为81.6%、42.9%,两者存在统计学差异(P0.05);结直肠癌组织最大Φ≥5cm结直肠癌tM2-PK阳性率高于最大Φ5cm结直肠癌,在TNM分期中Ⅲ+Ⅳ期tM2-PK阳性率高于Ⅰ+Ⅱ期,不同Duck分期上C+D期tM2-PK阳性率高于A+B期;在不同年龄、性别以及不同分化程度上tM2-PK阳性率无统计学差异(P0.05);40例结直肠癌患者癌组织tM2-PK阳性患者,平均评分7.53±1.94,其tM2-PK血清浓度26.59±10.24,两者呈正相关,r=0.876,P=0.001 5,有统计学意义(P0.05)。结论 tM2-PK在结直肠癌的早期诊断,病情评估以及预后判断上有广阔前景。     

双重实时荧光定量PCR检测产志贺毒素大肠埃希菌stx1和stx2基因

摘要：目的：建立一种检测产志贺毒素大肠埃希菌(STEC)的双重实时荧光定量PCR法。 方法：根据stx1和stx2及其变种序列,设计PCR引物和TaqMan探针,建立双重实时荧光定量PCR检测体系,进行灵敏度和特异性评价,并对STEC模拟粪便标本及动物粪便标本进行检测分析。 结果：双重实时荧光定量PCR检测含志贺毒素基因重组质粒的灵敏度为1×10² copies/反应体系;该法对17种常见肠道病原菌均无特异性扩增,对47株不同志贺毒素类型及变种的已知STEC菌株的检测特异性为100%;对模拟粪便标本的检测下限为3.55×10³ CFU/g;对动物粪便标本的检测阳性率高于细菌分离培养。 结论: 建立的双重实时荧光定量PCR可作为不同志贺毒素类型及变种的STEC菌株的快速鉴定方法,亦可用于人感染性腹泻标本和动物粪便标本STEC感染的快速筛查。     

联合检测肿瘤 M2型丙酮酸激酶、TPS 及 CEA 对结肠癌的诊断价值

目的：探讨联合检测肿瘤 M2型丙酮酸激酶（Tumor M2-PK）、组织多肽特异性抗原（TPS）及癌胚抗原（CEA）对结肠癌的诊断价值。方法收集74例结肠癌患者和60例健康志愿者分别检测血清 TPS、CEA 和粪便中 Tumor M2-PK 水平,对比分析 TPS、CEA 及 Tumor M2-PK 单一检测及联合检测的灵敏度、特异度及准确性。结果结肠癌观察组肿瘤标志物 TPS、CEA 及Tumor M2-PK 水平均明显高于健康对照组（P ＜0．05）。3种肿瘤标志物的灵敏度、特异度及准确性在单一检测及联合检测的差异均有统计学意义（P ＜0．05）。3种肿瘤指标单一检测、单一检测与联合检测之间比较,特异度及准确性均低于三者联合。结论肿瘤标志物 TPS、CEA 及 Tumor M2-PK 联合检测可提高对结肠癌诊断的灵敏度、特异度及准确性。     

细针穿刺标本M2型丙酮酸激酶基因检测在甲状腺乳头状癌诊断中的价值

目的 探讨细针穿刺标本M2型丙酮酸激酶基因检测在甲状腺乳头状癌诊断中的价值。方法 对行甲状腺结节细针穿刺检查的120例甲状腺结节患者(125个结节)应用细针穿刺采集标本,采用实时定量聚合酶链反应检测M2型丙酮酸激酶mRNA表达量,采用免疫组化技术检测术后组织中的M2型丙酮酸激酶蛋白表达。结果 本组120例患者共计125个结节,其中良性结节37个,甲状腺乳头状癌88个,甲状腺乳头状癌的M2型丙酮酸激酶mRNA表达量显著高于良性结节(P<0.05)。甲状腺乳头状癌的M2型丙酮酸激酶基因表达量在肿瘤最大直径方面存在显著差异(P<0.05),在年龄、性别、结节数目、BRAFV^600E基因型、淋巴结转移及美国癌症协会分期方面无统计学差异(P>0.05)。M2型丙酮酸激酶基因诊断甲状腺乳头状癌的特异度为95.7%,敏感度为46.9%,准确度为62.8%,阴性预测值为46.4%,阳性预测值为95.8%;联合检测M2型丙酮酸激酶基因、BRAFV^600E基因诊断甲状腺乳头状癌的敏感度、准确度显著高于单独检测M2型丙酮酸激酶基因、BRAFV     

人血浆DNA双重实时荧光定量PCR检测法的建立

目的 建立带有内参照的双重实时荧光定量PCR方法检测血浆DNA含量.方法 构建重组质粒DNA作为内参照物,采用共用下游引物的双重实时荧光定量PCR技术同步扩增人看家基因β-actin和重组质粒载体中人工合成DNA序列,定量检测健康成年人血浆DNA含量.结果 本法能在同一个反应管中对目的基因和内参照进行同步扩增,两者的扩增无相互干扰,特异性好;重组质粒DNA的平均扩增效率达90%,β-actin基因的平均扩增效率接近100%;本法批内变异系数（CV）11%,批间CV17%.结论 成功建立含有内参照的双重实时荧光定量PCR方法,可对血浆DNA进行定量检测.     

血浆肿瘤型M2丙酮酸激酶在直肠癌诊断与病情监测中的价值

目的探讨血浆肿瘤型M2丙酮酸激酶(TuM2-PK)水平在直肠癌患者中的变化,以及在直肠癌诊断与病情监测方面的价值。方法采用ELISA定量检测153例直肠癌患者、50例直肠良性肿瘤患者及32例健康对照者血浆TuM2-PK水平,比较3组间TuM2-PK的表达差异。以TuM2-PK15U/mL判断为阳性结果,分析不同临床病理参数直肠癌患者的TuM2-PK阳性率差异。ROC曲线法分析TuM2-PK诊断直肠癌TNM分期Ⅰ期、Ⅱ期的性能,Spearman分析血浆TuM2-PK与直肠癌各临床病理参数的相关性。结果直肠癌组、直肠良性病变组及健康对照组血浆TuM2-PK水平中位数分别为23.00、14.57、12.68U/mL,直肠癌组明显高于良性病变组及健康对照组(P0.05),良性病变组与健康对照组血浆TuM2-PK水平差异无统计学意义(P0.05)。直肠癌不同分化程度、TNM分期、是否存在淋巴结转移时的TuM2-PK表达阳性率存在明显差异;TuM2-PK诊断直肠癌及TNM分期Ⅰ期、Ⅱ期的曲线下面积分别为0.72、0.74、0.77;TuM2-PK水平与肿瘤细胞分化程度、淋巴结转移与否、TNM分期有关;血浆TuM2-PK检测直肠癌的灵敏度为62%,特异度为58%。结论血浆TuM2-PK变化与病情有一定相关性,对直肠癌的TNM分期、浸润转移的判断有帮助,可辅助诊断直肠癌。     

一种血浆肿瘤型M2-丙酮酸激酶检测方法——酶联免疫吸附试验的临床评价

目的评价一种血浆肿瘤型M2-丙酮酸激酶（TUM2-PK）检测方法——酶联免疫吸附试验（ELISA）的测定性能和临床适用性。方法对新的自主开发的TU M2-PK检测方法进行偏倚测定、不精密度测定、回收试验以及对371例临床标本进行测定。结果TU M2-PK检测方法有良好的准确度（偏倚〈5 U/mL）和精密度[变异系数（CV）〈10%]。371份临床标本的TUM2-PK水平测定结果与＂金标准＂病理结果对照,TUM2-PK的敏感性和特异性分别为69.7%（106/152）和86.8%（190/219）,准确度达到80.0%（296/371）。此外,该试剂盒所测得的恶性肿瘤患者的TU M2-PK水平明显高于良性肿瘤患者及正常对照者,差异具有统计学意义（P均〈0.01）。结论TU M2-PK ELISA测定性能良好,具有较好的临床应用价值。     

实时荧光定量PCR法与常规细菌鉴定法检测肠道致病菌的对比研究

目的探讨常规细菌鉴定法及实时荧光定量PCR法检测肠道致病菌的价值。方法就诊的3330例腹泻患者的粪便标本,同时进行常规细菌鉴定法及实时荧光定量PCR法检测,观察肠道致病菌中沙门菌及志贺菌检测阳性率。结果 3330例粪便标本中,常规细菌鉴定法检测沙门菌阳性率为3.90%(130/3330),志贺菌阳性率为1.65%(55/3330);实时荧光定量PCR法检测沙门菌阳性率为4.11%(55/3330),志贺菌阳性率为1.80%(60/3330),两种方法检测沙门菌及志贺菌阳性率比较,差异无统计学意义(P均0.05)。所有实时荧光定量PCR法检测阳性标本均经常规细菌鉴定确诊,阳性率为100%。结论实时荧光定量PCR法可直接从粪便中提取DNA,检测肠道致病菌,具有简便、快速等优点,大大缩短了检出时间,为临床早期诊断和治疗提供了依据,值得临床推广。     

血浆肿瘤型M2丙酮酸激酶与其他肺癌标志物联合检测对肺癌的诊断价值

目的 探讨血浆肿瘤型M2丙酮酸激酶(TuM2-PK)、神经元特异烯醇化酶（NSE）和细胞角蛋白19（CYFRA21-1）联合测定对肺癌的诊断价值。方法 用ELISA法分别检测肺癌患者组95例，良性肺部疾病组80例和健康体检组100名血中的TuM2-PK、NSE和CYFRA21-1的水平含量。结果 肺癌组3种指标水平均明显高于良性肺疾病组和健康对照组，差异有统计学意义（P<0.01）；TuM2-PK诊断肺癌的敏感度为66.3%，特异度为95%；TuM2-PK和NSE联合检测肺癌的阳性率最高为81%。结论 3项指标对肺癌辅助诊断均有一定的价值，TuM2-PK和NSE联合检测可以提高对肺癌的阳性诊断, TuM2-PK可作为一种新肺癌标志物应用。     

BioPlex 2200全自动免疫分析仪检测血清中抗双链DNA抗体的临床应用

目的分析比较BioPlex 2200全自动免疫分析仪和酶联免疫吸附测定(ELISA)检测血清中抗双链DNA(dsDNA)抗体的灵敏度、特异度及其优缺点。方法采用BioPlex 2200和ELISA检测101例纳入研究者的血清抗dsDNA抗体。纳入研究者中49例为临床确诊的系统性红斑狼疮患者,52例作为对照(包括32例其他结缔组织病患者和20例体检健康者)。结果BioPlex 2200检测抗dsDNA抗体的灵敏度为57.14%,特异度为94.23%;ELISA检测抗dsDNA抗体的灵敏度为53.06%,特异度为96.15%。两种方法总体符合率为95.05%,χ2检验分析,差异无统计学意义(χ2=78.69,P>0.05),Kappa值评价一致性较好(Kappa=0.880)。BioPlex 2200检测抗dsDNA抗体,3组抗dsDNA抗体水平比较,差异有统计学意义(F=61.52,P<0.05)。结论BioPlex 2200和ELISA检测血清抗dsDNA抗体结果有较好的一致性,检测迅速,值得临床推广应用。     

Tumor M2 pyruvate kinase: a tumor marker and its clinical application in gastrointestinal malignancy

Proliferating cells, in particular tumor cells, express a dimeric isoenzyme of pyruvate kinase, termed Tumor M2 pyruvate kinase. In the last few years, much attention has been paid to this novel tumor marker that can be determined in EDTA-plasma and in the feces. It has been used in diagnosis and surveillance of a variety of malignant diseases. As compared with the established tumor markers, Tumor M2-PK in EDTA-plasma proves to have at least equal sensitivity in pancreatic, gastric, esophageal, colorectal and cholangiocellular cancer. In combination with established tumor markers, EDTA-plasma M2-PK is a useful tool in diagnosis and surveillance of gastrointestinal tumors. In colorectal cancer, M2-PK in EDTA-plasma even proves superiority as compared with CEA. Fecal Tumor M2-PK testing resembles a good noninvasive screening parameter for colorectal cancer with a reported sensitivity of 68.8-91.0% and a specificity of 71.9-100%. It is superior to fecal occult blood testing in colorectal cancer screening. Since it is effective, easy to handle and bears rather low costs, fecal Tumor M2-PK testing is recommended for large-scale CRC screening.     

A novel method to capture methylated human DNA from stool: implications for colorectal cancer screening

BACKGROUND: Assay of methylated DNA markers in stool is a promising approach for colorectal cancer (CRC) screening. A method to capture hypermethylated CpG islands from stool would enrich target analyte and allow optimal assay sensitivity. METHODS: Methyl-binding domain (MBD) protein was produced using a pET6HMBD plasmid with MBD DNA sequence cloned from rat MeCP2 gene and bound to a column of nickel-agarose resin. We first established the feasibility of using the MBD column to extract methylated human DNA in a high background of fecal bacterial DNA. To explore the impact of MBD enrichment on detection sensitivity, the tumor-associated methylated vimentin gene was assayed with methylation-specific PCR from stools to which low amounts of cancer cell DNA (0-50 ng) were added and from stools from CRC patients and healthy individuals. Stools from cancer patients were selected with low amounts of human DNA (median 7 ng, range 0.5-832 ng). RESULTS: With MBD enrichment, methylated vimentin was detected in stools enriched with >/=10 ng of cancer cell DNA and in CRC stool with a range of native human DNA amounts from 4 to 832 ng. Without MBD enrichment, methylated vimentin was not detected in the enriched stools and was detected in only 1 cancer stool with high human DNA (832 ng). In stools from healthy individuals methylated vimentin was not detected, with or without MBD enrichment. CONCLUSIONS: MBD capture increases assay sensitivity for detecting methylated DNA markers in stool. Applied clinical studies for stool cancer screening are indicated.     

肿瘤型M2丙酮酸激酶与癌类抗原19-9联合检测在胰腺癌诊断中的应用

目的:探讨肿瘤型M2丙酮酸激酶(TumorM2-PK,TuM2-PK)、糖类抗原19-9(CA19-9)在胰腺癌中的表达特性。方法:采用ELISA法分别检测50例胰腺癌患者、35例胰腺炎患者及80名健康体检人血中TuM2-PK、CA19-9含量。结果:胰腺癌患者血中TuM2-PK、CA19-9值分别是(164.9±41.3)U/mL、(630.1±200.3)U/mL。在胰腺癌的诊断中单独检测时TuM2-PK、CA19-9敏感性分别为75%、74.5%,特异性分别为90%、85%,联合测定敏感性、特异性分别达到82.3%、100%。结论:联合检测TuM2-PK、CA19-9有助于提高对胰腺癌诊断的特异性和敏感性。     

Plasma levels of tumor M2-pyruvate kinase should not be used as a tumor marker for hematological malignancies and solid tumors.

It has been reported that the dimeric isoform of the enzyme pyruvate kinase M2 was overexpressed in various solid tumor cells. Hence, it was suggested that circulating levels of the so-called tumor M2-pyruvate kinase (Tu M2-PK) could be used as a tumor marker for monitoring systemic therapies of various solid tumors. We analyzed its validity as a tumor marker by comparing plasma levels of Tu M2-PK in patients with different non-malignant diseases to levels in healthy individuals and in patients with hematological diseases. Plasma levels of Tu M2-PK were measured using an ELISA assay in a total of 284 patients. The mean Tu M2-PK concentration of 32 U/mL was significantly higher in the group of patients with hematological malignancies (n = 121) (p < 0.001). However, 37% of healthy individuals (n = 63) and 44% of patients with non-malignant diseases (n = 100), especially patients with an acute inflammatory reaction (67%), were found to have elevated levels of Tu M2-PK using a cutoff level of 15 U/mL. The specificity was 59% and the sensitivity was 51%. There was no significant correlation between the prevalence of a hematological malignancy and positive Tu M2-PK result. Thus, our data imply that Tu M2-PK is not a useful tumor marker for hematological malignancies and solid tumors, as a significant number of false positive results were detected in healthy individuals and patients with non-malignant diseases.     

类风湿关节炎五项诊断指标的应用价值评价

目的 评价分析抗瓜氨酸化波形蛋白(MCV)抗体、抗葡萄糖-6-磷酸异构酶(GPI)抗体等5项指标在类风湿关节炎(RA)患者血清中的表达与疾病的相关性及应用价值.方法 收集150例BA、32例SLE、30例强直性脊柱炎(OA)、20例骨关节炎(AS)、20例干燥综合征(SS)、20例结缔组织病(CTD)患者和40名健康人血清标本.采用ELISA分析血清中抗MCV、抗GPI和抗环瓜氨酸肽(CCP)抗体水平,采用间接免疫荧光法检测抗角蛋白(AKA)抗体,采用速率散射比浊法检测类风湿因子(RF)水平.分别计算和比较单项标志物诊断RA的效能;选择高敏感度与高特异度抗体联合检测,并计算其与RF联合诊断RA的效能.结果 5种抗体在RA患者组中的阳性率显著高于疾病对照组和健康对照组(X2分别为88.5、76.0、279.2、88.2、94.8,P均＜0.05).除AKA抗体阳性率较低(37.3%)外,其余抗体在RA中的阳性率差异无统计学意义(X2分别为21.9、9.4、20.2、43.2、41.6,P均＞0.05).单项抗体检测中,抗MCV和抗GPI抗体的敏感度最高,分别为78.0%和83.3%,抗CCP抗体的特异度最高,为97.1%,具有高特异度(96.1%)的AKA抗体敏感度最低,仅为49.4%.抗MCV/抗CCP抗体联合检测的敏感度和特异度最高,分别为92.7%和96.9%.RF/抗GPI/抗CCP抗体联合检测的敏感度和特异度为90.7%和96.9%,RF/抗MCV/抗CCP抗体联合检测的敏感度和特异度为94.0%和96.9%.结论 抗MCV和抗GPI抗体诊断RA的敏感度最高;抗CCP抗体的特异度最高,AKA抗体虽然具有较高的特异度,但敏感度最低,联合检测可弥补单项抗体检测造成的RA漏诊,RW抗GPI/抗CCP抗体和RE/抗MCV/抗CCP抗体的组合模式可同时提高诊断RA的敏感度和特异度.