用多重及套式PCR同时检测血清中的HBV和HCV方法的建立

首次应用多重及套式ＰＣＲ技术同时检测人血清中的ＨＢＶ和ＨＣＶ。将抽提的ＨＢＶＤＮＡ和（或）ＨＣＶＲＮＡ在含ＡＭＶ逆转录酶、ＴａｑＤＮＡ多聚酶以及ＨＢＶ和ＨＣＶ外套引物的ＰＣＲ缓冲引物的ＰＣＲ缓冲液中进行逆转录后连续进行ＰＣＲ扩增，以第一轮扩增产物为模板，在含ＨＢＶ和ＨＣＶ内套引物的ＰＣＲ反应体系中进行第二轮扩增，产物经电泳后，以ＤＮＡ分子量标志物或已知片段做参考，出现５２３ｂｐ和（或）２６０ｂｐ产     

孕妇血清和胚胎组织中微小病毒B19 DNA的检出

作者建立了检测人微小病毒Ｂ１９（ＨＰＶ－Ｂ１９）的ＰＣＲ方法。两对引物（Ｐ１Ｐ２和Ｐ３Ｐ４）的设计均位于编码ＨＰＶ－Ｂ１９衣壳蛋白ＶＰ１基因区内，扩增产物分别为７００ ｂｐ和１０４ ｂｐ。比较Ｐ１Ｐ２－ＰＣＲ和Ｐ３Ｐ４－ＰＣＲ，二者均具有高度特异性和第三性，ＨＰＶ－Ｂ１９ ＤＮＡ最小检同量分别为５ ｆｇ。应用Ｐ１Ｐ２－ＰＣＲ对３２份孕妇血清和２１份胚胎组织标本进行了ＨＰＶ－Ｂ１９ ＤＮＡ的检测，结     

HBV－DNA套式一次PCR方法的建立

应用乙型肝炎病毒ＤＮＡＣ基因区的一对外引物（ＨＢＣ＿１和ＨＢＣ＿２，扩增片段长度为５６６ｂｐ）及一对内引物（ＨＢＣ＿３和ＨＢＣ＿４，扩增片段长度为３０１ｂｐ）对不同稀释度的ＨＢＶ－ＤＮＡ（ａｄｒ亚型）进行套式一次ＰＣＲ扩增，结果表明灵敏度可达到１０ａｇ水平。此方法快速、简便、灵敏、材异，与分子杂交的灵敏度相当。     

HBV—DNA套式一次PCR方法的建立

应用乙型肝炎病毒ＤＮＡ Ｃ基因区的一对外引物（ＨＢＣ１和ＨＢＣ２，护增片段长度为５６６ ｂｐ）及一对内引物（ＨＢＣ３和ＨＢＣ４，护增片段长度为３０１ ｂｐ）对不同稀释度的ＨＢＶ－ＤＮＡ（ａｄｒ亚型）进行套式一次ＰＣＲ扩增，结果表明灵敏度可达到１０ａｇ水平。此方法快速、简便、灵敏、特异，与分子杂交的灵敏度相当。     

人血小板生成素在大肠杆菌中的高效表达

目的：人血小板生成素（ＴＰＯ）在大肠杆菌中的高效表达。方法：利用ＰＣＲ技术,扩增出了（１－１７４个氨基酸的）人血小板生成素ｃＤＮＡ,并将其克隆到ｐＧＥＸ－１λＴ载体中,构建了融合蛋白表达质粒。重组子用限制性内切酶酶切鉴定。表达产物ＳＤＳ－聚丙烯胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）。结果：限制性内切酶酶切鉴定表明重组子含有正确的（１－１７４氨基酸）ＴＰＯｃＤＮＡ片段;表达产物经ＳＤＳ－ＰＡＧＥ相对分子质量     

创伤后多器官功能不定综合征与HLA—DRB1基因的相关性研究

目的 研究ＨＬＡ－ＤＲＢ１基因与创伤后多器官功能不全综合征（ＭＯＤＳ）的相关性。方法 采用多聚酶链反应－序列特异性引物分析法（ＰＣＲ－ＳＳＰ）,对３７例创伤后ＭＯＤＳ患者ＨＬＡ－ＤＲＢ１基因进行分型,用酶联免疫吸附试验（ＥＬＩＳＡ）进行血浆白细胞介素－１０（ＩＬ－１０）测定,并与相应健康人５２例结果比较。结果 创伤后ＭＯＤＳ患者ＨＬＡ－ＤＲＢ１＊１５０１￣ＤＲＢ１＊１５０２（编码ＨＬＡ－ＤＲ２抗原     

抗结肠癌抗体重链可变区和κ轻链基因的克隆

目的：从一株抗结肠癌细胞系ＬＳ１７４Ｔ的单抗细胞株ＣＬ－４中克隆出抗结肠癌抗体重链可变区和κ轻链基因。方法：用一步法提取总ＲＮＡ，逆转录形成ｃＤＮＡ，设计并合成一套扩增小鼠抗体重链可变区和完整κ轻链的通用引物，通过ＰＣＲ扩增基因，分别克隆入ｐＵＣ１９，作核苷酸序列分析。结果：用重链引物扩增获得长约３６０ｂｐ的片段；用轻链引物扩增获得约６４０ｂｐ的片段，序列测定获得它们的ＤＮＡ序列。结论：克隆的重链可变区基因长度为３６０ｂｐ，属于小鼠重链可变区Ｉ（Ｂ）亚族；κ轻链长度为６４２ｂｐ，其中可变区为３２１ｂｐ，属于小鼠κ轻链Ⅴ亚族。     

改进的锚定反转录PCR法克隆中国人庚型肝炎病毒3′末端基因

应用改进的锚定反转录ＰＣＲ（ａｎｃｈｏｒｅｄＲＴ－ＰＣＲ）法，在总ＲＮＡ的３′端“加尾”，Ｏｌｉｇｏｔｅｘ提纯ｐｏｌｙ（Ａ）＋ｍＲＮＡ，Ｏｌｉｇｏｄ（Ｔ）－ａｎｃｈｏｒ引物合成第１链ｃＤＮＡ，ａｎｃｈｏｒ引物及特异引物进行“半巢式”ＰＣＲ，从献血员血清中扩增出ＨＧＶ３′末端基因片段。序列分析结果表明：此中国株ＨＧＶ３′末端基因序列与美国株存在较大差异，但阅读框架相似。用此方法克隆单链ＲＮＡ病毒基因组的３′末端。由于许多动物病毒和９０％以上的植物病毒均为单链ＲＮＡ基因组，故该技术也可应用于其它病毒基因组未知末端的基因克隆。     

聚合酶链反应快速诊断白色念珠菌菌血症

目的：应用聚合酶链反应（ＰＣＲ）技术诊断白色念珠菌菌血症。方法：设计的引物特异性扩增编码白色念珠菌细胞色素Ｐ４５０Ｌ１Ａ１的基因，其长度为２４３ｂｐ。ＥＤＴＡ抗凝血用去污剂溶解，用ＤＮａｓｅＩ去除人体白细胞和污染的细菌ＤＮＡ；念珠菌的细胞壁用溶胞酶消化并用ＳＤＳ和蛋白酶Ｋ裂解。用酚／氯仿／异戊醇提取模板ＤＮＡ。用ＰＣＲ技术扩增。结果：与细菌、病毒及人体细胞ＤＮＡ无非特异扩增。检出血中白色葡萄的阈值     

利用T─载体克隆法对人脑源性神经营养因子全长基因PCR产物的克隆

利用Ｔ－载体克隆法完成了含信号肽及前导肽的人脑源性神经营养因子（ｂｒａｉｎ－ｄｅｒｉｖｅｄｎｅｕｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ，ＢＤＮＦ）全长基因ＰＣＲ产物的克隆。序列测定结果表明，所克隆的人ＢＤＮＦ基因从起始密码子ＡＴＧ到终止密码子ＴＡＧ的７７４ｂｐ中，除下游端ＰＣＲ引物因为采用猪的ＰＣＲ引物而有一个碱基改变外，其它序列与国外文献报道序列完全一致。该碱基改变不影响氨基酸序列     

Validity of apparent diffusion coefficient (ADC) value in diagnosis and follow-up of multiple sclerosis patients in different clinical subtypes

Background

Multiple sclerosis (MS) is a chronic disease with a wide range of pathologic changes that modify the apparent diffusion coefficient (ADC) value.

D-二聚体、纤溶酶原激活物抑制物在急性肾衰竭患者血液净化中的临床意义

目的探讨急性肾衰竭(ARF)时D-二聚体(D-D)、纤溶酶原激活物抑制物(PAI)在不同的血液净化方法中的动态变化及临床意义。方法对我院2007年1月—2010年3月急诊住院58例ARF患者血液净化前、净化后4 h及38例健康人进行血浆中D-D含量及PAI水平测定。ARF患者血液净化方法随机采用血液透析(HD)(31例)、血液透析滤过(HDF)(27例)。结果 ARF患者D-D含量及PAI水平较对照组明显升高[D-D(0.83±0.04)与(0.48±0.03)mg/L,P=0.000 2;PAI(14.95±0.73)与(8.03±0.30)103kat/L,P<0.0001;]HD治疗4 h后D-D含量和PAI活性较治疗前升高[D-D(0.89±0.05)与(1.48±0.37)mg/L,P=0.018;PAI(14.89±1.78)与(22.10±3.56)103kat/L,P=0.025],而HDF治疗后D-D含量和PAI无明显变化[D-D(0.91±0.06)与(1.12±0.09)mg/L,P=0.65;PAI(15.81±1.98)与(16.10±2.56)103kat/L,P=0.86]。结论 ARF患者D-D、PAI水平升高,存在凝血-纤溶系统的紊乱,HD可以加重这种改变,而采用HDF治疗可避免对患者凝血机能的影响,在急性肾衰竭治疗中有一定临床价值。     

Role of multidetector computed tomography (MDCT) angiography in preoperative assessment of coarctation of the aorta in pediatric patients and young adults

IntroductionAim of this work is to assess the reliability of 64-slice multidetector computed tomographic (MDCT) angiography for the preoperative assessment of coarctation of the aorta in pediatric patients and young adults.Material and methodsTwenty eight patients with clinical suspicion of coarctation of the aorta who underwent both Doppler echocardiography and MDCT angiography were included in the study. MDCT angiography findings were compared with both Doppler echocardiography and surgical results.ResultsThe overall sensitivity of three-dimensional MDCT angiography for diagnosis of the coarctation of the aorta was (100%) which was higher than that of Doppler echocardiography (91%).The overall sensitivity of MDCT angiography for the assessment of cardiac defects was (88%) which was lower than that of Doppler echocardiography (100%).ConclusionWe concluded that MDCT angiography with multiplanar and three dimensional techniques can be considered the modality of choice for preoperative assessment of coarctation of the aorta in pediatric patients and young adults.     

Percutaneous radiofrequency ablation of hepatocellular carcinoma in the presence of portal vein thrombosis

Portal vein thrombosis (PVT) may not be an absolute contraindication for hepatic radiofrequency ablation (RFA). Although the data are sparse, PVT is commonly considered a contraindication to RFA. PVT has actually been described as a complication following RFA. RFA was used to treat a 3.9×2.9 cm primary hepatocellular carcinoma (HCC) in a patient with concomitant PVT without complication. RFA can be safely performed in this setting but further studies could clarify this issue.     

Influence of maturational status in the exercise-induced release of cardiac troponin T in healthy young swimmers

ObjectivesTo determine the influence of maturational status on the release of cardiac troponin T (cTnT) induced by a bout of 30 min, high-intensity, continuous exercise.DesignQuasi-experimental, cross-sectional study.MethodsSeventy male, young, well trained swimmers (age range 7–18 years, training experience 1–11 years) were classified by maturational stages: Tanner stage I (n = 14), II (n = 15), III (n = 15), IV (n = 13), and V (n = 13). Participants underwent a distance-trial of 30 min continuous swimming, and cTnT was measured before, immediately after and 3 h after exercise. Changes in cTnT over time were compared among groups, and associated with exercise load.ResultsBasal cTnT was higher in Tanner-V (3.8–8.1 ng/L) compared with I (1.5–5.5 ng/L, p < 0.001), II (1.5–4.5 ng/L, p < 0.001) and III (1.5–6.8 ng/L, p = 0.003), and in IV (1.5–6.3 ng/L) compared with II (p = 0.036). Maximal elevations of cTnT from baseline were notable (p < 0.001) and comparable among maturational stages (p = 0.078). The upper reference limit for myocardial injury was exceeded in 35.7% of the participants, without differences among groups (p = 0.18). Baseline cTnT correlated with participant characteristics, and maximal cTnT elevations from baseline with exercise internal load (%HRpeak, r_s = 0.34, p =  0.003; %HRmean, r_s = 0.28, p = 0.02).ConclusionsMaturational status influences positively absolute pre- and post-exercise cTnT but not its elevation after a bout of 30 min, high-intensity, continuous exercise.     

Application of selective saturation to image the dynamics of arterial blood flow during brain activation using magnetic resonance imaging.

A saturation-based approach is proposed to image the arterial blood flow signal with temporal resolution of 1 to 2 s and in-plane spatial resolution of a few millimeters. Using a saturation approach to suppress the undesired background stationary signal allows the blood water that enters the slice to be imaged at some specified later time. Since the blood protons that are being imaged are not restricted to the intravascular space, this technique is also sensitive to tissue perfusion signal contributions. The signal uptake characteristics of the saturation method proposed were used to study the different signal contributions as a function of the acquisition parameters. A typical perfusion acquisition (FAIR) was also used for comparison. The proposed method was demonstrated in a functional motor activation experiment and the observed signal changes were smaller than those obtained using the FAIR acquisition. The dynamics of the saturation method and FAIR temporal signal changes were investigated and time constants between 2 and 44 s were estimated. The tissue signal contribution to the saturation method's signal was small over the range of acquisition parameters that sensitized it to the arterial compartment.     

128-multidetector CT: For assessment of optimal depth of electrode array insertion in cochlear implant operations

Objective

To assess the diagnostic reliability of MDCT in pre-operative evaluation of cochlear implant candidates and post-operative, estimation of depth of insertion.

磁共振扩散峰度成像预测食管癌放疗敏感性的实验研究

目的 从动物模型层面探讨扩散峰度成像（DKI）在预测食管癌放疗敏感性方面的应用价值。方法 建立人食管癌Eca-109裸鼠移植瘤模型,实验组给予单次剂量15 Gy（6 MV X射线）照射,对照组不接受任何处理。比较两组移植瘤体积及表观弥散系数（ADC）、平均扩散峰度（MK）、平均扩散系数（MD）的变化情况,观察实验组与对照组在相应时间点的细胞密度和坏死比例。结果 实验组裸鼠移植瘤在照射后明显出现了生长延迟现象,从照后第7天开始实验组移植瘤体积明显小于对照组（t=3.206~6.149,P<0.05）。照后第3天开始实验组ADC值及MD值明显高于对照组,MK值明显低于对照组（t_ADC=-11.018~-2.049,t_MD=-6.609~-2.052,t_MK=2.492~9.323,P<0.05）。照后第3天开始对照组细胞密度高于实验组,实验组的坏死比例高于对照组（t_密度=-8.387~-2.239,t_坏死比例=2.980~17.430,P<0.05）。结论 单次大剂量照射可以抑制荷瘤裸鼠肿瘤生长,ADC、MK、MD值均可以先于肿瘤形态学变化而发生改变,移植瘤细胞密度及坏死比例的变化与ADC、MK、MD值的变化基本吻合。DKI具有早期预测食管癌放疗敏感性的价值。     

高压氧辅助治疗对卒中后抑郁患者血清5-HT、NE及神经功能的影响

目的：观察高压氧（HBO）辅助治疗对卒中后抑郁（PSD）患者血清5-羟色胺（5-HT）、去甲肾上腺素（NE）及神经功能的影响。方法70例PSD患者根据数字表法随机分为2组,对照组（n=35例）采用常规措施治疗,观察组（n=35例）待确定活动性出血已稳定或已趋于稳定后,在对照组治疗基础上加用HBO治疗。两组疗程均为30d,比较两组患者治疗前后血清5-HT、NE表达水平及汉密尔顿抑郁量表（HAMD）、中国脑卒中量表（CSS）,改良Barthel指数（MBI）评分变化。结果两组患者治疗后血清5-HT、NE表达水平均明显升高（P<0.05）,且观察组升高较对照组更为显著（P<0.05）;两组患者治疗后HAMD、CSS评分明显降低（P<0.05）,而MBI评分明显升高（P<0.05）,且观察组降低或升高较对照组更为显著（P<0.05）。结论 HBO辅助治疗可明显升高PSD患者血清5-HT、NE表达水平,改善抑郁状态和神经功能。     

The role of magnetic resonance imaging in early detection of recurrent breast cancer

Purpose

The purpose of this work was the evaluation of new advances of magnetic resonance imaging (MRI) in diagnosis of recurrent breast cancer after conservative surgery, chemotherapy and radiotherapy.