胃癌淋巴结转移灶与原发灶P-糖蛋白的表达及意义

目的:探讨P-糖蛋白(P-glycoprotein,P—gp)在胃癌淋巴结转移灶与原发灶表达水平的差异及其与胃癌临床病理特征的关系.方法:应用免疫组化的方法检测19例伴有淋巴结转移的胃癌患者的淋巴结转移灶、胃癌原发灶和正常胃黏膜的P—gp表达.结果:淋巴结转移灶P-gp表达的阳性率高于原发灶(84.20％ vs 52.63％,P<0.05);淋巴结转移灶P-gp表达与患者性别、年龄、肿瘤分化程度和浸润深度无关;胃癌原发灶P-gp的表达与胃癌组织分化程度和浸润深度有关(P<0.05),与性别和年龄无关.P-gp表达阳性率在高、中分化者较低分化者高,肿瘤浸润未达浆膜者高于已穿越浆膜者.结论:胃癌淋巴结转移灶的P-gP表达高于原发灶,淋巴结转移灶与原发灶P-gp表达和胃癌临床病理特征的关系不同.     

结肠癌转移淋巴结P-gp、Bcl-2、Bax表达与体外化疗敏感性的关系

目的 探讨结肠癌淋巴结转移灶多药耐药相关因子P-gp、Bcl-2、Bax表达及其与肿瘤细胞体外化疗敏感性的关系.方法 对新鲜结肠癌肿瘤组织及转移淋巴结进行肿瘤细胞培养体外化疗药敏性试验,并对原发灶和转移灶行P-gp、Bcl-2、Bax免疫组化染色.结果 ①P-gp、Bax在转移灶中的表达程度明显高于原发灶(均P<0.05),而Bcl-2在原发灶中表达强于转移灶(P<0.01).在原发灶与转移淋巴结间P-gp、Bax表达均具有明显相关性 (r=0.660 6、0.399 5,均P<0.01).②在结肠癌原发灶中Bcl-2与Bax表达呈正相关(r=0.305 1,P<0.05).③10种化疗药物中5-FU、VP-16、THP、MMC对转移淋巴结肿瘤细胞的抑制率高于原发灶;HCPT、L-OHP、CDDP、VCR对原发灶肿瘤细胞抑制率高于转移灶(均P<0.05).④P-gp表达程度在原发灶与5-FU、VCR、PTX、VP-16的肿瘤细胞抑制率呈负相关,在转移灶则与PTX、eADM呈负相关;Bcl-2在原发灶表达强度与5-FU、PTX、MMC的抑制率呈负相关,在转移淋巴结中则与HCPT、PTX、L-OHP、eADM的抑制率均具有负相关性;Bax表达强度在原发灶与5-FU、VP-16对肿瘤细胞的平均抑制率呈正相关,在转移灶则随CDDP、L-OHP、eADM抑制率升高而增强(均P<0.05).结论 结肠癌淋巴结转移灶中耐药相关因子的表达及化疗药敏性均呈现与原发灶不同的异质性,术后辅助化疗应针对淋巴结转移灶进行.     

老年胃癌患者区域转移淋巴结中COX-2与多药耐药因子相关性分析

目的探讨老年患者胃癌原发灶、区域淋巴结转移灶中环氧合酶-2(COX-2)与多药耐药因子P-糖蛋白(P-gp)、谷胱甘肽S转移酶-π(GST-π)及DNA拓扑异构酶Ⅱα(TopoⅡα)表达的关系及意义。方法免疫组化染色法检测33例伴区域淋巴结阳性转移的老年胃癌患者(≥60岁)标本中COX-2、P-gp、GST-π、TopoⅡα的表达情况,同法检测32例对照组(<60岁)胃癌原发灶、转移灶中4种蛋白的表达,比较其在两组患者之间、淋巴结转移灶与原发灶之间的强度表达差异,并进行相关分析。结果老年患者P-gp在转移灶表达高于原发灶(P<0.05),TopoⅡα则在转移灶中表达降低(P<0.05);TopoⅡα表达在原发灶、转移灶间存在正相关关系(r=0.499 3,P<0.01)。老年患者原发灶组织中COX-2与TopoⅡα之间存在负相关表达(r=-0.588 9,P<0.01),P-gp与TopoⅡα之间存在正相关表达(r=0.382 0,P<0.05);转移灶各因子表达间未发现相关关系(均P>0.05)。原发灶老年组COX-2、P-gp和GST-π表达明显高于对照组(均P<0.05);转移灶老年组COX-2、P-gp的表达高于对照组(均P<0.05)。结论老年胃癌患者与对照组比较,COX-2及部分多药耐药因子存在异质性表达,老年患者多药耐药性与其他年龄患者不同,对老年患者进行多药耐药性逆转的胃癌生物治疗应根据COX-2、多药耐药因子的表达特点进行。     

老年贲门癌组织中caspase-3表达及其临床意义

目的研究半胱氨酸天冬氨酸蛋白酶3(caspase-3)表达在老年贲门癌发生发展中的作用。方法采用免疫组化S-P法观察113例老年贲门癌旁上皮细胞、原发癌细胞和浸润淋巴细胞以及转移灶癌细胞中caspase-3表达,并分析原发灶癌细胞caspase-3表达与贲门癌临床病理特征的关系。结果caspase-3在贲门癌旁上皮细胞中的表达高于原发灶癌细胞(P<0·05);caspase-3在贲门癌原发灶浸润淋巴细胞中的阳性率高于贲门癌旁上皮细胞和原发灶癌细胞(P<0·05);转移灶癌细胞中caspase-3阳性率高于原发灶癌细胞(P<0·05);原发灶癌细胞中caspase-3表达与贲门癌患者的年龄、性别以及贲门癌肿瘤大小、浸润深度、转移、TNM分期、生长方式和分化程度无相关性(P<0·05)。结论贲门癌原发灶癌细胞caspase-3表达下调和浸润淋巴细胞caspase-3表达上调与贲门癌发生有关,来自原发灶和转移微环境中的化学物质可能通过提高转移灶癌细胞中caspase-3表达,进而起到抑制转移灶的作用。     

结直肠癌组织中耐药基因P-gp、GST、TopoⅡ的表达

目的分析耐药基因P-gp(P-糖蛋白)、GST-π(谷胱甘肽S-转移酶)及TopoⅡ(DNA拓扑异构酶Ⅱ)在结直肠癌组织中的表达,并探讨其临床病理意义。方法采用免疫组化SP法(链霉菌抗生物素蛋白-过氧化物酶连结法)检测74例结直肠癌组织中P-gp、GST-π及TopoⅡ的表达情况,并分析其与结直肠癌临床分期、组织类型、肿瘤大小、浸润程度、淋巴结转移及预后等之间的关系。结果本组74例患者中P-gp阳性62例(83.78%),TopoⅡ阳性60例(81.08%),GST-π阳性70例(94.59%)。P-gp表达与患者年龄、性别、分化程度无明显相关性(P0.05),但与淋巴结转移、浸润程度有关,发生淋巴结转移患者阳性率明显高于未转移者(P0.05);TopoⅡ表达仅与淋巴结转移情况有关,而结直肠癌组织中GST-π表达与患者年龄、性别、转移及浸润程度等临床病理特征无关(P0.05)。结论 P-gp、GST-π及TopoⅡ耐药基因的表达与结直肠癌患者多药耐药密切相关,化疗治疗前,对患者P-gp、GST-π及TopoⅡ的检测对化疗药物的选择、化疗效果及预后具有指导意义。     

胃癌组织中Kiss-1 mRNA与Notch1 mRNA的表达及意义

目的探讨Kiss-1 mRNA与Notch1 mRNA在胃癌组织中的表达及意义。方法采用实时荧光定量聚合酶链反应检测胃癌和癌旁正常组织中Kiss-1 mRNA与Notch1 mRNA的表达情况,分析其与临床病理特征之间的关系。结果 Kiss-1 mRNA在胃癌组织中的表达较癌旁正常组织中降低(P0.05);Kiss-1 mRNA的表达与胃癌的淋巴结转移、TNM分期密切相关(P0.05),与胃癌的浸润深度、分化程度、远处转移无关(P0.05);Notch1 mRNA在胃癌组中的表达较癌旁正常组织升高(P0.05);Notch1 mRNA的表达与胃癌的淋巴结转移、TNM分期、分化程度密切相关(P0.05),与胃癌的浸润深度、远处转移无关(P0.05)。结论 Kiss-1在胃癌的发病过程中发挥抑癌基因作用,并通过抑制转移来影响胃癌的预后;Notch1在胃癌的发病过程中发挥癌基因作用,有潜力成为早期诊断胃癌的新型肿瘤标记物。     

结直肠癌组织中Lgr5、GATA6表达变化及临床意义

目的 探讨结直肠癌（CRC）组织中富含亮氨酸重复单位的G蛋白耦联受体5(Lgr5)和转录调节因子GATA结合蛋白6(GATA6)的表达变化及临床意义。方法 选取手术切除CRC组织41例为实验组,其配对的癌旁正常组织（距离癌组织边缘>5cm）作为对照组。采用免疫组化法检测组织中Lgr5、GATA6表达,分析两者与结直肠癌临床病理特征的关系,Spernman秩相关分析两者的相关性。结果 CRC组织及癌旁正常组织中Lgr5蛋白高表达率分别为60.98%(25/41)、12.20%(5/41),比较差异有统计学意义（P<0.05）;CRC组织及癌旁正常组织中GATA6蛋白高表达率分别为63.41%(26/41)、21.95%(9/41),比较差异有统计学意义（P<0.05）。Lgr5蛋白高表达率与CRC的TNM分期、淋巴结转移有关（P均<0.05）,与患者性别、年龄、肿瘤部位、肿瘤大小、分化程度和浸润深度无关（P均>0.05）。GATA6蛋白高表达率与CRC的浸润深度、淋巴结转移有关（P均<0.05）,与患者性别、年龄、肿瘤部位、肿瘤大小、分化程度和肿瘤分期...     

P-糖蛋白表达与中国食管癌临床病理特征相关性Meta分析

目的探讨P-糖蛋白(P-gp)在中国食管癌组织中的表达及与食管癌临床病理特征的相关性。方法检索数据库,查找国内外2000～2016年发表的关于P-gp表达及其与食管癌临床病理特征相关性文献,采用RevM an5. 3软件进行Meta分析。结果 P-gp在癌组织中表达:Meta分析纳入6篇文献,含食管癌469例,癌旁组织251例,食管癌与癌旁组织P-gp表达差异有统计学意义[OR=3. 65,95%CI(2. 51,5. 30),P=0. 000];与淋巴结转移相关性:Meta分析纳入11篇文献,含食管癌823例,在无、有淋巴结转移组中P-gp表达差异有统计学意义[OR=0. 64,95%CI(0. 47,0. 87),P=0. 005];与肿瘤浸润深度相关性:Meta分析纳入7篇文献,含食管癌565例,浸润黏膜层、黏膜下层和肌层与浸润浆膜层P-gp表达差异有统计学意义[OR=0. 55,95%CI(0. 37,0. 81),P=0. 003];与肿瘤分化程度相关性:Meta分析纳入11篇文献,含食管癌796例,P-gp在高、中分化组与低分化组表达差异无统计学意义[OR=0. 79,95%CI(0. 54,1. 15),P=0. 220]。结论 P-gp在食管癌组织中高表达,且与淋巴结转移、浸润深度密切相关,与肿瘤分化程度无关,P-gp的表达可作为评估食管癌淋巴结转移和浸润深度的重要指标。     

胃癌组织中NPRL2 mRNA的表达变化及意义

目的观察胃癌组织中NPRL2 mRNA的表达变化,并探讨其临床意义。方法采用逆转录聚合酶链反应(RT-PCR)法检测60例胃腺癌组织及其癌旁正常组织中NPRL2 mRNA的表达。结果 NPRL2 mRNA在胃癌及癌旁正常组织中均有表达,但胃癌组织中NPRL2 mRNA的表达显著降低(IOD比值为1.099±0.285),与癌旁正常组织(IOD比值为1.582±0.305)相比P<0.05。NPRL2 mRNA的表达与肿瘤分化程度、有无淋巴结转移、肿瘤浸润深度、临床分期及患者的年龄、性别无显著相关(P均>0.05)。NPRL2 mRNA在不同分化程度、不同临床分期、有无淋巴结转移及不同年龄、性别患者的胃癌组织中的表达无显著差异(P均>0.05)。结论胃癌组织中NPRL2mRNA表达降低,其在胃癌的发生、发展过程中可能发挥重要作用。     

MACC1、HGF、c-Met蛋白在胃癌组织中的表达及意义

目的探讨胃癌组织中结肠癌转移相关基因-1(metastasis-associated in colon cancer-1,MACC1)、肝细胞生长因子(hepatocyte growth factor,HGF)、肝细胞生长因子受体(mesenchymal epithelial transition factor,c-Met)的表达与临床病理参数的关系及其意义。方法应用免疫组化方法检测129例胃癌患者手术切除的癌组织及癌旁正常组织中MACC1、HGF、c-Met的表达情况,分析其与胃癌临床病理参数之间的关系及在胃癌组织中表达的相关性。结果 MACC1、HGF、c-Met在胃癌组织中的表达显著高于癌旁正常组织(P0.05)。MACC1在胃癌组织中的表达与性别、年龄、肿瘤大小、肿瘤部位、有无淋巴结转移、有无腹膜转移、总生存时间(OS)、无病生存时间(DFS)无关(P0.05),而与肿瘤浸润深度、TNM分期、组织分化程度显著相关(P0.05)。HGF在胃癌组织中的表达与性别、年龄、肿瘤部位、有无腹膜转移无关(P0.05),而与肿瘤大小、浸润深度、有无淋巴结转移、TNM分期、组织分化程度、OS(5年)、DFS(5年)显著相关(P0.05)。c-Met在胃癌组织中的表达与性别、年龄、肿瘤大小、肿瘤部位、有无腹膜转移无关(P0.05),而与肿瘤浸润深度、有无淋巴结转移、TNM分期、组织分化程度、OS(5年)、DFS(5年)显著相关(P0.05)。在胃癌组织中,MACC1与HGF的表达呈正相关(r=0.182,P0.05),MACC1与c-Met的表达呈正相关(r=0.508,P0.05),HGF与c-Met的表达呈正相关(r=0.523,P0.05)。结论 MACC1、HGF、c-Met在胃癌组织中的表达显著升高,三者与胃癌的发生、发展密切相关,可能协同参与胃癌的进程。     

妊娠时间与肺结核复发的相关性研究及诊治对策

目的 分析肺结核史患者妊娠时间和肺结核复发间相关性.方法 选取我院收治的有肺结核史的妊娠妇女576例作为研究对象,对其妊娠前肺结核治疗、治愈后妊娠时间、妊娠后复发肺结核等进行分析,总结有肺结核史育龄女性的妊娠时间和肺结核复发之间的关系.结果 肺结核治愈后不同时间段妊娠者的结核复发率比较,差异具有显著性（P＜0.05）,停药后间隔时间越久妊娠,肺结核复发的几率越小.结论 加强孕期痰菌检查,及早发现复发肺结核,提高母婴安全.     

我国骨关节结核诊治的回顾与展望

骨关节结核是危害人们健康的严重感染性疾病,近95％由他处结核病继发而来.罹患骨关节结核疾病后几乎均将致残,严重影响人们的健康、工作和生活.建国以来在党和国家的关心和支持下,骨关节结核的诊治水平取得了长足进步.时至今日,由于多种原因,学科发展和被重视程度受到一定的制约,同整个医疗行业的发展不相适应.回顾过去,展望未来,我们需要重新审视骨关节结核的诊治方法,努力推进骨关节结核诊疗技术的科学发展.     

Effect of phosphorylation of MAPK and Stat3 and expression of c-fos and c-jun proteins on hepatocarcinogenesis and their clinical significance

AIM To study the effect of phosphorylation ofMAPK and Stat3 and the expression of c-fos andc-jun proteins on hepatocellular carcinogenesisand their clinical significance.METHODS SP immunohistochemistry was usedto detect the expression of p42/44~(MAPK), p-Stat3,c-fos and c-jun proteins in 55 hepatocellularcarcinomas (HCC) and their surrounding livertissues.RESULTS The positive rates and expressionlevels of p42/44~(MAPK), p-Stat3, c-fos and c-junproteins in HCCs were significantly higher thanthose in pericarcinomatous liver tissues (PCLT).A positive correlation was observed between theexpression of p42/44~(MAPK) and c-fos proteins, andbetween p-Stat3 and c-jun, but there was nosignificant correlation between P42/44~(MAPK) and p-Stat3 in HCCs and their surrounding livertissues.CONCLUSION The abnormalities of Ras/Raf/MAPK and JAKs/ Stat3 cascade reaction maycontribute to malignant transformation ofhepatocytes. Hepatocytes which are positive forp42/ 44~(MAPK), c-fos or c-jun proteins may bepotential malignant pre-cancerous cells.Activation of MAPK and Stat3 proteins may be anearly event in hepatocellular carcinogenesis.     

Overweight and Obesity and Progression of ADPKD

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Effect of phosphorylation of MAPK and Stat3 and expression of c-fas and c-jun proteins on hepatocarcinogenesis and their clinical significance

AIM To study the effect of phosphorylation ofMAPK and Stat3 and the expression of c-fos andc-jun proteins on hepatocellular carcinogenesisand their clinical significance.METHODS SP immunohistochemistry was usedto detect the expression of p42/44MAPK, p-Stat3,c-fos and c-jun proteins in 55 hepatocellularcarcinomas (HCC) and their surrounding livertissues.RESULTS The positive rates and expressionlevels of p42/44MAPK, p-Stat3, c-fos and c-junproteins in HCCs were significantly higher thanthose in pericarcinomatous liver tissues (PCLT).A positive correlation was observed between theexpression of p42/44MAPK and c-fos proteins, andbetween p-Stat3 and c-jun, but there was nosignificant correlation between p42/44MAPK and p-Stat3 in HCCs and their surrounding livertissues.CONCLUSION The abnormalities of Ras/Rat/MAPK and JAKs/ Stat3 cascade reaction maycontribute to malignant transformation ofhepatocytes. Hepatocytes which are positive forp42/ 44MAPK, c-fos or c-jun proteins may bepotential malignant pre-cancerous cells.Activation of MAPK and Stat3 proteins may be anearly event in hepatocellular carcinogenesis.     

Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice

Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10h postinjection in experiment 1 and about 4h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5alpha-pregnane-3beta,11beta,21-triol-20-one (measuring corticosterone metabolites with a 5alpha-3beta,11beta-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals' sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology.     

Replication and Inhibitors of Enteroviruses and Parechoviruses

The Enterovirus (EV) and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV). They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.     

荣宝和氯硝柳胺灭螺效果比较及成本分析

目的 评价新型灭螺药物荣宝杀灭钉螺的效果,探讨其推广应用价值.方法 按目前推荐的荣宝灭螺剂量,喷洒法为30 g/m2,浸杀法为50 g/m3;氯硝柳胺喷洒法和浸杀法分别采用2 g/m2和2 g/m3杀螺剂量,分别在室内和现场进行灭螺试验,观察两种药物的灭螺效果并初步分析评估其成本.结果 在现场气温22～30℃条件下,荣宝50 g/m3浸杀3、5、7 d后,螺袋内钉螺校正死亡率均达到100.0%,与氯硝柳胺2 g/m3灭螺效果相似;荣宝30 g/m2剂量喷洒3、5、7、15 d后,钉螺校正死亡率分别为54.5%、58.0%、69.0%、79.1%,氯硝柳胺喷洒组钉螺校正死亡率分别为61.0%、69.4%、76.7%、77.9%.在室温18℃条件下,荣宝以30 g/m2喷洒3、5、7、15 d后,钉螺校正死亡率分别为72.9%、87.2%、91.5%、76.1%;而相应2 g/m2氯硝柳胺喷洒后的钉螺校正死亡率分别为81.3%、95.7%、97.9%、80.4%.同样完成1000 m2的喷洒灭螺任务,荣宝所需灭螺药物和人力资费成本比氯硝柳胺多支出0.114元/m2;完成72 m3的浸杀灭螺任务,荣宝所需灭螺药物和人力资费成本比氯硝柳胺多支出0.127元/m3.50 g/m3荣宝浸杀灭螺剂量,对成鱼(＞250 g)的活力不会造成影响,但对鱼类幼苗仍具较强毒性.结论 荣宝与氯硝柳胺灭螺效果相似,由于其成本较高,氯硝柳胺仍然是目前首选灭螺药物,但荣宝的鱼类毒性低,可作为氯硝柳胺之外有益的补充灭螺药物.     

心电图、心电向量图与冠状动脉造影结果对比分析

目的:通过分析心电图(Electrocardiogram,ECG)和心电向量图(Vectorcardiogram,VCG)的改变与冠脉造影（CAG）结果进行对比,探讨ECG、VCG在冠状动脉病变中的诊断价值。方法: 选择2008年1月~2009年12月临床拟诊断为冠心病患者108例,行常规ECG、VCG检查,并于1周内进行CAG,对检查结果依据各自的诊断标准进行判定,以CAG为标准诊断法,利用四格表法,计算相关评价真实性的指标并进行比较。结果: ①VCG检测的灵敏度、特异度、准确度显著高于ECG(P<0.05,P<0．01)。②ECG、VCG阳性率与冠脉病变支数组间比较:在单支病变、双支病变中,VCG阳性率明显高于ECG(P<0.05),左主干或三支病变无统计学意义;组内比较:ECG组左主干或三支病变组较单支病变、双支病变阳性率高(P<0.05,P<0.01);VCG组左主干或三支病变组较单支病变阳性率高(P<0.05);与双支病变阳性率比较无统计学意义;③ECG、VCG阳性率与冠脉病变程度组间比较:冠脉病变狭窄50%～69%的VCG阳性率明显高于ECG (P<0.05),其他两组阳性率比较无统计学意义;组内比较:ECG组冠脉病变狭窄≥90%较50%～69%、70%～89%的阳性率高(P<0.05,P<0.01); VCG组狭窄≥90%较50%～69%阳性率高（P<0.01),其他无统计学意义。结论: VCG对冠心病检测价值显著高于ECG。     

Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O(2)(.-)). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E(+)). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and O(2)(.-) formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the O(2)(.-) that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E(+). However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of O(2)(.-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O(2)(.-). Analysis of the fluorescence characteristics of ethidium (E(+)) and 2-OH-E(+) strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O(2)(.-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable [corrected] for detecting intracellular O(2)(.-).