TORCH综合征与先天性畸形

近年来,先天性感染引起新生儿畸形死亡的人数日益增加, Ｔ Ｏ Ｒ Ｃ Ｈ 是一组能引起先天性感染的病原微生物。１９７１ 年 Ｎａｈｍ ｉａｓ 等将引起先天性感染的病原体用英文字头命名,称为 Ｔ Ｏ Ｒ Ｃ Ｈ 感染或 Ｔ Ｏ Ｒ Ｃ Ｈ 综合征。 Ｔ 代表弓形体（ Ｔｏｘｏｐｌａｓｍ ａ ｇｏｎｄｉｉ, Ｔ Ｏ Ｘ Ｏ）, Ｏ为其它（ Ｏｔｈｅｒ,包括很多种病毒）, Ｒ 表示风疹病毒（ Ｒｕｂｅｌｌａ ｖｉｒｕｓ）, Ｃ代表巨细胞病毒（ Ｃｙｔｏｍ ｅｇａｌｏｖｉｒｕｓ, Ｃ Ｍ Ｖ）, Ｈ 表示单纯疱疹病毒（ Ｈｅｒｐｅｓ ｓｉｍ ｐｌｅｘ ｖｉｒｕｓ, Ｈ Ｓ Ｖ）。随着病原微生物学、免疫学和流行性病学研究的进展,又发现了多种能引起先天性感染的病原微生物,如水痘--带状疱疹病毒（ Ｖａｒｉｃｅｌｌａ ｚｏｓｔｅｒ ｖｉｒｕｓ, Ｖ Ｚ Ｖ）,麻疹病毒（ Ｍｅａｓｌｅｓ ｖｉｒｕｓ, Ｍ Ｖ）, 流 行性腮 腺炎 病毒（ Ｐａｒｏｔｉｔｉｓ ｖｉｒｕｓ, Ｐ Ｖ）, 人类免 疫缺 陷病毒 （ Ｈｕｍ ａｎｉｍ ｍ ｕｎｏｄｅｆｉｃｉｅｎｃｙ ｖｉｒｕｓ, Ｈ Ｉ Ｖ）,丙型肝炎病毒（ Ｈｅｐａｔｉｔｉｓ Ｃ ｖｉｒｕｓ, Ｈ Ｃ Ｖ）,人类疱疹病毒６型（ Ｈｕｍ ａｎ ｈｅｒｐｅｓ ｖｉｒｕｓ ｔｙｐｅ ６, Ｈ Ｈ Ｖ ６）,肠道病毒（ Ｅｎｔｅｒｏｖｉｒｕｓ, Ｅ Ｖ）,乙型肝炎病毒（ Ｈｅｐａｔｉｔｉｓ Ｂ ｖｉｒｕｓ, Ｈ Ｂ Ｖ）,人类乳头瘤病毒（ Ｈｕｍ ａｎ ｐａｐｉｌｌｏｍ ａｖｉｒｕｓ, Ｈ Ｐ Ｖ）, Ｅ Ｂ 病毒（ Ｅｐｓｔｅｉｎ Ｂａｒｒ ｖｉｒｕｓ, Ｅ Ｂ Ｖ）,人类微小病毒 Ｂ１９（ Ｈｕｍ ａｎ ｐａｒｖｏｖｉｒｕｓ Ｂ１９, Ｈ Ｐ Ｖ Ｂ１９）和人类嗜血细胞病毒１ 型（ Ｈ Ｔ Ｌ Ｖ １）等。 Ｔ Ｏ Ｒ Ｃ Ｈ 感染的特点是孕妇患其中任何一种疾病后,本人的症状极其轻微,或根本没有症状和特征。病原体虽不相同,却能使胎儿或新生儿出现相同或相似的临床表现,有时还很严重,甚至导致死亡。如在怀孕早期感染,则发生流产、死胎和胎儿畸形。中晚期感染,导致胎儿不同程度畸形和脏器损害。     

抗ToRCH—IgM型抗体临床对比检测研究

ＴｏＲＣＨ系列病原微生物（包括弓形虫Ｔｏｘ、风疹病毒ＲｕＶ、巨细胞病毒ＣＭＶ、单纯疱疹病毒ＨＳＶ　　Ⅰ／Ⅱ等）是一组具有致畸作用的病原体,孕妇感染可导致流产　、早产、畸形甚至死胎。目前ＴｏＲＣＨ　ＩｇＭ型抗体的检测都在不同程度上受到非特异性的干扰,采用有效方法消除干扰对ＴｏＲＣＨ　ＩｇＭ型抗体检测的准确性至关重要。为此我们对比了多种消除ＩｇＭ检测中非特异性干扰　的方法,并以最佳方法对２５６例女性献血者、１４３例正常妊娠和６１例异常妊娠标本进行ＴｏＲＣＨ　　ＩｇＭ抗体的对比检测。结果显示健康人群中ＴｏＲＣＨ活动性感染率较高,健康孕妇ＴｏＲＣＨ　ＩｇＭ的　总阳性率高达２３．１％（其中Ｔｏｘ５．６％、ＣＭＶ９．８％、ＲｕＶ８．４％、ＨＳＶ４．９％）;而异常妊娠则显著上升　（　ｐ＜０．０１）,其中Ｔｏｘ、ＣＭＶ、ＲｕＶＩｇＭ阳性率上升明显（ｐ＜０．０５）,分别为１９．７％、２６．２％、２４．６％　。　由此可见孕期ＴｏＲＣＨ病原微生物活动感染是致异常妊娠的主要因素之一,应严密监视孕期ＴｏＲ　ＣＨ病原微生物活动性感染,特别是弓形虫、风疹病毒和巨细胞病毒的感染。     

青心酮对妊娠高血压综合征胎盘血管内皮和平滑肌细胞内一氧化氮合酶的影响

本文对正常孕妇、妊娠高血压综合征（ＰＩＨ）患者和经青心酮（ＤＨＡＰ）治疗的ＰＩＨ患者等共２４例,应用组织化学分析方法观察胎盘血管内皮细胞（ＶＥＣ）和平滑肌细胞（ＶＳＭＣ）内一氧化氮合酶（ＮＯＳ）活性的变化。结果表明：正常孕妇胎盘ＶＥＣ和ＶＳＭＣ内ＮＯＳ活性较高;ＰＩＨ胎盘ＶＥＣ和ＶＳＭＣ内ＮＯＳ活性明显减弱,并伴有组织和细胞的形态学损伤;经ＤＨＡＰ治疗后的ＰＩＨ胎盘ＶＥＣ和ＶＳＭＣ细胞ＮＯＳ活性较未经ＤＨＡＰ治疗者明显增加,其组织和细胞损伤也减轻。本研究结果提示胎盘内ＶＥＣ和ＶＳＭＣ细胞的ＮＯＳ减少可能与ＰＩＨ的发生和／或发展有关,青心酮治疗ＰＩＨ的作用可能与ＤＨＡＰ促进胎盘ＶＥＣ和ＶＳＭＣ内一氧化氮（ＮＯ）合成有关。     

人巨细胞病毒PP71基因序列分析与表达

人巨细胞病毒 (Ｈｕｍａｎｃｙｔｏｍｅｇａｌｏｖｉｒｕｓ)在人群中存在非常普遍 ,大多数呈临床不显性或潜伏感染 ,孕妇ＨＣＭＶ复发感染或新的感染均可引起新生儿宫内或围产期感染 ,导致胎儿畸形、智力低下和发育迟缓等。人是ＨＣＭＶ的唯一宿主 ,病毒可通过人与人间的直接或间接接触传播。近年来对ＨＣＭＶ的致病机理的研究已日趋深入 ,已有多项研究证实 ,ＨＣＭＶ ＰＰ71(ＵＬ82 )基因是病毒抗原之一。亦有研究证实 ,ＨＣＭＶ ＰＰ71是 (ＵＬ82 )基因产物 ,可促进病毒后期的基因表达 ,提高病毒的复制能力。本实验对ＰＰ71(ＵＬ82 )基因…     

脂多糖对血管平滑肌细胞一氧化氮合酶基因表达及细胞增殖的影响

应用ＲＮＡ印迹分析和亚硝酸盐含量测定检查脂多糖（ＬＰＳ）对大鼠血管平滑肌细胞（ＶＳＭＣ）一氧化氮合酶（ＮＯＳ）基因表达及ＮＯ合成的影响,用３Ｈ－ＴｄＲ参入实验观察ＬＰＳ对细胞ＤＮＡ合成的影响．结果表明,ＬＰＳ在诱导ＶＳＭＣｉＮＯＳｍＲＮＡ表达和促进ＮＯ合成的同时,抑制ＶＳＭＣＤＮＡ合成．证明ＬＰＳ的作用与其浓度和作用时间有关     

慢性高山病低氧通气反应降低与肺功能缺损

对１４例慢性高山病（ＣＭＳ）患者的低氧肺泡通气不足的发病机理和肺功能作了研究。与对照组比较,ＣＭＳ组的ＰＥＴＣＯ２高,ＶＴ低,低氧通气反应（ＨＶＲ）Ａ值低;吸入高浓度Ｏ２后,ＣＭＳ已降低的ＨＶＲ。ＣＭＳ的ＦＥＶ１／ＶＣ比值降低并加重动脉低氧血症。结果表明：周围性ＨＶＲ降低和中枢性低氧通气抑制是引起高原低氧肺泡通气不足的因素。而肺泡通气不足与阻塞性肺疾病是导致ＣＭＳ发生的主要原因。     

脂多糖对血管平滑肌细胞一氧化氮合酶基因表达及细胞增殖的影响

应用ＲＮＡ迷分析和亚硝酸盐含量测定检查脂多糖（ＬＰＳ）对大鼠血管平滑肌细胞（ＶＳＭＣ）一氧化氮合酶（ＮＯＳ）基因表达及ＮＯ合成的影响,用Ｔ３Ｈ－ＴｄＲ参入实验观察ＬＰＳ对细胞ＤＮＡ合成的影响,结果表明,ＬＰＳＤ 诱导ＶＳＭＣｉＮＯＳｍＲＮＡ表达和促进ＮＯ合成的同时,抑制ＶＳＭＣＤＮＡ合成,证明ＬＰＳ的作用与其浓度和作用时间有关。     

天然和氧化修饰型LDL、VLDL及HDL对培养人动脉平滑肌细胞原癌基因fos及myc表达的影响

动脉平滑肌细胞（ＳＭＣ）是动脉粥样硬化（ＡＳ）斑块中的主要细胞,它的增殖在ＡＳ形成过程中极其重要。脂蛋白和氧化修饰型脂蛋白对ＳＭＣ增殖的影响以及ＳＭＣ增殖与原癌基因异常表达的关系是当前ＡＳ发病机制研究的热点之一。我们在建立人主动脉ＳＭＣ体外培养方法的基础上,观察了ＬＤＬ,ＶＬＤＬ及ＨＤＬ和相应的氧化修饰型脂蛋白对培养人ＳＭＣｆｏｓ,ｍｙｃ,ｅｒｂ－Ｂ原癌基因转录表达的影响。结果表明：①ＨＤＬ对ＳＭＣｆｏｓ,ｍｙｃ基因表达无影响;②ＬＤＬ和ＶＬＤＬ有使这些基因表达增加的趋势,但与对照比较差异不显著（Ｐ＞０．０５）;③ＯＸ－ＶＬＤＬ,ＯＸ－ＶＬＤＬ和ＯＸ－ＨＤＬ有使ＳＭＣｆｏｓ,ｍｙｃ基因表达显著增强的作用（Ｐ＜０．０１）,且其作用较相应的天然脂蛋白大（Ｐ＜０．０１）．上述结果说明：ＬＤＬ,ＶＬＤＬ,ＯＸ－ＬＤＬ,ＯＸ－ＶＬＤＬ和０Ｘ－ＨＤＬ的致ＡＳ作用可能与刺激ＳＭＣｆｏｓ和ｍｙｃ癌基因表达增加有关。     

MAPK对胰岛素介导的人血管平滑肌细胞PKCα的影响

目的：在胰岛素的干预下,观察ＭＡＰＫ反义寡核苷酸（ＯＤＮｓ）对人血管平滑肌细胞（ＶＳＭＣ）增殖及ＰＫＣα表达的影响。方法：３ＨＴｄＲ掺入法检测ＶＳＭＣ增殖,逆转录ＰＣＲ、免疫组织化学法检测ＰＫＣα表达。结果：反义ＯＤＮｓ 处理的细胞可显著抑制胰岛素诱导的ＶＳＭＣ的ＤＮＡ合成,ＯＤＮｓ 的上述作用与降低ＶＳＭＣ内ＰＫＣα基因表达有关。结论：胰岛素刺激人ＶＳＭＣ增殖可被ＭＡＰＫ反义寡核苷酸所抑制,可能存在有关胰岛素ＰＫＣＭＡＰＫ激活途径     

MnSOD与CuZnSOD基因转染细胞在电离辐射敏感性中的作用

诱导超氧阴离子（Ｏ２·）的产生是电离辐射损伤细胞和组织的重要机制之一。超氧化物歧化酶ＭｎＳＯＤ和ＣｕＺｎＳＯＤ是细胞内清除Ｏ２·的主要抗氧化酶。本研究将ＭｎＳＯＤ和ＣｕＺｎＳＯＤｃＤＮＡ真核表达载体分别导入ＣＨＯ细胞,以稳定表达这两种酶的ＣＨＯ细胞系为细胞模型,通过分析细胞形态学、线粒体功能和受照射细胞Ｄ０值变化比较研究了ＭｎＳＯＤ和ＣｕＺｎＳＯＤ对细胞辐射敏感性的影响。结果表明,ＭｎＳＯＤ表达的增强能明显降低细胞的辐射敏感性,同时维持线粒体的正常代谢功能,而ＣｕＺｎＳＯＤ则不能。由此提示ＭｎＳＯＤ和ＣｕＺｎＳＯＤ对细胞的辐射敏感性存在着不同的调控机制。     

疱疹病毒定量PCR的建立

A single pair of oligonucleatide primer selected within a highly conserved region of the DNA polymerase gene in herpesviruses was synthesized. The competitive template DNA purified from cytomegalovirus (CMV) DNA was used to carry out competiitve PCR amplification with herpes simplex virus type 1 (HSV1) DNA (target sequences). And anti-HSV1 effects of acyclovir (ACV) was investigated by the method.The results showed that the efficacy of PCR amplification was equal to each other(the ratio of the quantity of c…     

Development and Padronization of Three Multiplex PCRs for the Diagnosis of Chlamydia trachomatis, Toxoplasma gondii, Herpes Simplex Viruses 1 and 2, and Cytomegalovirus

To develop multiplex PCRs (mPCRs) that allows simultaneous diagnosis of the infectious agents Chlamydia trachomatis, Toxoplasma gondii, HSV 1/2, and Cytomegalovirus (CMV). The study included patients with clinical suspicion of these agents, and clinical samples were blood, cerebrospinal fluid, urine, vaginal swabs, and amniotic fluid. After the extraction of DNA, this was used as a template in amplification by PCR of selected genes. The following conditions were tested: primer concentration, MgCl₂ concentration, and annealing temperature. Three mPCRs were developed: multiplex I (CMV, HSV 1/2), multiplex II (CMV, HSV 1/2, T. gondii), and multiplex III (C. trachomatis, T. gondii, HSV 1/2, and CMV). The primer pairs used were shown to be specific for each infectious agent, and the specificity of mPCR assays was 100 %. Both the reactions of the monoplex PCR and mPCR produced a detection limit of 2 × 10^?5 to 6 × 10^?7 ng/μl of different DNAs. Upon conclusion, amplified products of expected size were obtained in 3 different reactions, and all the infectious agents were detected simultaneously in each mPCR. The concordant results of the study suggest that mPCR can be a powerful tool to improve the diagnostics of infectious diseases.     

单纯疱疹病毒的PCR直接基因分型

本文利用ＰＣＲ技术建立一种对ＨＳＶ直接基因分型的方法。在ＨＳＶ－Ⅰ、Ⅱ两型的ＤＮＡ多聚酶基因上设计了一条两型共同的上游引物（ＨＤＰ－Ｂ）和两条型特异的下游引物（ＨＤＰ－１、ＨＤＰ－２）。三条引物共同组成一个扩增反应体系,在ＨＳＶ－Ⅰ产生５４３ｂｐ条带,ＨＳＶ－Ⅱ产生３７２ｂｐ条带,据此在基因水平上对ＨＳＶ进行分型。５株不同来源的ＨＳＶ（２株Ⅰ型,３株Ⅱ型）分型结果与病毒分离及血清学方法完全一致。该反应体系与其它来源的ＤＮＡ不产生特异反应,敏感性可达１ｆｇ。应用该法对１５１份临床可疑ＨＳＶ感染的标本进行检测并分型,结果与免疫学方法完全一致。     

Utilization of herpes simplex PCR assays for cerebrospinal fluid in a pediatric health care setting

An assessment was made of the utilization and impact of a diagnostic polymerase chain reaction (PCR) assay for the diagnosis of herpes simplex viruses (HSV) 1 and 2 in cerebrospinal fluid of children who attended a Canadian pediatric referral centre. One hundred and three assays were performed on specimens from 103 patients during the period August 1997 to September 1998. Patient ages ranged from newborn to 16 years. Indications for HSV PCR included seizures with or without fever (56.3%), aseptic meningitis (16.5%), and encephalopathy with or without fever (10.7%). Only 2 of 103 (1.9%) assays were positive, including one each for HSV1 and HSV2. Control specimens that were seeded with virus indicated inhibition for 24.3, 8.8, and 6.8% of assays for HSV1, HSV2, and both HSV1 and HSV2, respectively. The mean turn-around time for HSV PCR was 2.5 days, and 90.3% were completed in less than 5 days. Acyclovir was administered to 78.6% of the patients overall; the results of the HSV PCR impacted on the treatment courses for 36 individuals. Nevertheless, 16.5% of patients continued to receive extended courses of antiviral therapy despite negative HSV PCR assays. Although it is desirable to decrease the frequency of PCR inhibitions and to further decrease the interval to assay completion, HSV PCR does have a significant impact on antiviral use in this setting.     

Comparison of primers for the detection of Salmonella enterica serovars using real-time PCR

AIMS: To evaluate the specificity and sensitivity of PCR primers for the detection of Salmonella enterica in a real-time PCR assay using pure cultures. METHODS AND RESULTS: Unenriched whole cells in sterile water were used as template for each PCR. SYBR Green dye was used for the nonspecific detection of dsDNA. The real-time PCR detection limits of five previously published primer sets used in conventional PCR applications were not below 3 x 10(3) CFU per reaction (rxn). A new primer set, Sen, was designed, which detected Salm. enterica Newport down to 6 CFU rxn(-1) in one case, and gave an average detection limit of 35 CFU rxn(-1) over three separate runs. CONCLUSIONS: Primers originally designed for end-point PCR did not have adequate specificity or sensitivity compared with those specifically designed for real-time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study emphasizes the importance of evaluating real-time PCR primer sets in pure cultures prior to testing in field samples. This study will benefit other researchers in selecting an appropriate primer set for real-time PCR detection of Salm. enterica.     

Polymerase chain reaction for identification of herpes simplex virus (HSV-1), cytomegalovirus (CMV) and human herpes virus-type 6 (HHV-6) in oral swabs

Considering that sensitive and specific methods to detect HSV-1, CMV and HHV-6 on oral mucosa have a great impact on oral diagnosis practice and research, together with the evidence that PCR is a rapid and reliable method, the purpose of the present study was to develop primer sets to detect HSV-1, CMV and HHV-6 in oral swabs by nested polymerase chain reaction (nested PCR). We developed a practical method for sample collection without tissue trauma, and the swabs were stored until used for DNA extraction. After the nested PCR a DNA fragment of 241 bp corresponding to HSV-1 was amplified. DNA fragments of 224 and 369 bp were amplified corresponding to CMV and HHV-6, respectively. DNA sequencing analysis confirmed the expected sequences of each virus. In conclusion, it was demonstrated that these new primer sets are able to identify HSV-1, CMV and HHV-6 in oral swab using nested PCR.     

痘病毒科不同病毒属特异的PCR检测方法的建立

目的:建立可准确、快速地鉴别诊断可感染人的不同属痘病毒的特异PCR方法。方法:设计针对正痘病毒属、副痘病毒属和传染性软疣病毒属的多对特异引物,并制备相应的DNA模板,针对不同的模板优化引物与反应条件,分别进行检测筛选,建立病毒属特异的单独与多重PCR方法。结果:单一模板的PCR扩增反应中,正痘病毒的检测敏感性可达101拷贝/μL(引物为OPEaL-F1880/OPEaL-R2057),副痘病毒的检测敏感性可达101拷贝/μL(引物为PP2/PP3),传染性软疣病毒的检测敏感性为100 pg/μL体系(引物为MCV1/MCV2);混合模板的PCR扩增反应中,各属特异的引物均可获得预期大小的特异片段。结论:我们建立的PCR诊断方法,可用于痘病毒科不同病毒属感染的实验室特异快速鉴别诊断。     

桥式PCR，一种简易连接DNA标签序列的方法

MAST方法采用人工文库的DNA标签序列鉴定mRNA的可接近位点。大量的标签序列通过扩增和克隆测序达到阐明mRNA结合位点图。设计了单一引物的PCR,其引物在标签序列两端结合搭桥,在扩增中DNA标签序列在搭桥引物的作用下进行连接,连接的标签序列再克隆和测序。十几条这样的连接产物包含了上千条标签序列。该PCR方法简单、高效以用于高通量的方式对标签序列测序。     

On-chip PCR amplification of very long templates using immobilized primers on glassy surfaces

In this paper we describe a novel method for visualizing very long DNA fragments (for example >6 kb) which are difficult to spot with commonly used arrayers or capillary samplers with very small nanoliter volumes, using directly bound primers on "on-chip" polymerase chain reaction (PCR). We have used the genomes of the M13 bacteriophage (7.2 kb) the human mitochondrion (16.5 kb) as examples of long DNA templates to test the PCR and were able to elicit robust reactivity. Over 75% of the immobilized primers could be elongated to their fullest extent. In addition we were able to elicit the PCR reaction with double stranded templates in which one primer was immobilized and the other suspended in the reaction solution. These synthesized PCR products were visualized by either confocal microarray scanning or fluorescence microscopy using Cy5-dye fluorescence of the modified free primer, or the fluorescence of intercalating dyes.