肝组织中HBV cccDNA荧光定量聚合酶链反应检测法的建立

目的 建立和优化一种灵敏、特异的检测肝组织中乙型肝炎(乙肝)病毒(HBV)共价闭合环状DNA(cccDNA)荧光定量聚合酶链反应(荧光定量PCR).方法 设计检测HBV DNA(tDNA)和cccDNA特异性引物及探针,用3.44×100-3.44×109copies/μl含HBV全基因组序列(C基因型)质粒作为标准品,建立荧光定量PCR检测标准曲线.取33例乙肝肝癌患者肝组织标本、13例慢性乙肝患者肝活检组织标本和10例非乙肝患者肝组织标本,验证该法灵敏度和特异度.提取肝组织中DNA,取一部分进行质粒安全ATP依赖的DNA酶(PSAD)酶切;另一部分不酶切作为tDNA和β-globin检测样本,分别进行HBV cccDNA、tDNA和p-globin定量检测,以β-globin为参比,对每个细胞的HBV cccDNA和tDNA含量进行标准化.结果 检测肝组织中HBV cccDNA和tDNA定量的线型范围均为3.44 × 100-3.44×109 copies/μl.检测HBV cccDNA和HBV DNA下限均为3.44×100copies/μl.33例乙肝肝癌患者和13例慢性乙肝患者的肝组织中HBV cccDNA最低含量分别为0.003 copies/cell和0.031 copies/cell;检测10份非乙肝肝癌患者的肝组织标本均为阴性.应用PSAD消化肝组织中提取的DNA可减少假阳性,提高cccDNA检测法特异度达7.24× 102倍.对2例乙肝肝癌患者的肝组织标本重复检测5次,Ct值的变异系数为0.224%～0.609%.结论 该方法灵敏度和特异度高,重复性好,可用于检测肝组织中HBV cccDNA.

Abstract：

Objective To establish and optimize a sensitive and specific quantitative realtime polymerase chain reaction(PCR)method for detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA)in liver tissue. Methods Specific primers and probes were designed to detect HBV DNA(tDNA)and cccDNA. A series of plasmids(3.44 × 100-3.44 × 109 copies/μl)containing a full double-stranded copies of HBV genome(genotype C)were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma(HCC), 13 Chronic hepatitis B patients(CHB)and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase(PSAD)for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount(copies/cell)of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. Results The standard curves of real-time PCR with a linear range of 3.44 × 100 to 3.44 × 109 copies/μl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 100 copies/μl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 102 times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficience of variations on cycle threshold (Ct)were between 0.224%-0.609%. Conclusion A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.     

树立医院精神增强医院活力

新世纪医院的改革发展，已不完全是技术、设备这些有形资源的竞争，更重要的是医院文化的竞争。现代文化已成为一个医院、一个行业发展的战略资源，要打造出一个优秀的、具有实力的现代品牌医院，必需要营造构建一个良好的医院精神风貌，增强医院活力，以此提高医院的竞争力。医院精神，是医院文化、医院价值观的集中体现，是医院形象的灵魂，是医院最宝贵     

肝组织中HBV cccDNA荧光定量聚合酶链反应检测法的建立

Objective To establish and optimize a sensitive and specific quantitative realtime polymerase chain reaction(PCR)method for detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA)in liver tissue. Methods Specific primers and probes were designed to detect HBV DNA(tDNA)and cccDNA. A series of plasmids(3.44 × 100-3.44 × 109 copies/μl)containing a full double-stranded copies of HBV genome(genotype C)were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma(HCC), 13 Chronic hepatitis B patients(CHB)and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase(PSAD)for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount(copies/cell)of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. Results The standard curves of real-time PCR with a linear range of 3.44 × 100 to 3.44 × 109 copies/μl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 100 copies/μl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 102 times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficience of variations on cycle threshold (Ct)were between 0.224%-0.609%. Conclusion A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.     

构建癌症患者家属培训机构的必要性、可行性及策略分析——基于患者的知识和技能需求

从国内安宁疗护现状出发,指出建立癌症患者家属培训机构的必要性、可行性;从家庭照顾的基础护理知识、与患者疾病相关的知识和心理方面分析癌症患者家属的需求;根据癌症患者家属的需求,借鉴国内外累积的经验,从政府、医院和家属个人层面提出对策,旨在为构建符合社会需求的癌症患者家属培训机构提供参考。     

2台血细胞计数仪测定结果的比对研究

目的：探讨科室2台血细胞计数仪测定结果的可比性。方法：依据NCCLS EP9-A2文件,每天选取患者新鲜全血标本8份,分别用比较系统Sysmex XT-2000i和实验系统Abbott CD1700这2台血细胞计数仪重复测定标本2次,共测定5 d,应用Excel软件对所测结果进行统计。结果：CD1700与XT-2000i血细胞计数仪测定WBC、RBC、Hb和PLT的相关系数（r）分别为0.997 3、0.998 9、0.997 0和0.998 6,二者的相关性很好（r〉0.975）;对于临床可接受性评价,除了RBC不可接受外,其余3项的预期偏倚均未超过1/2CLIA′88。结论：2台血细胞计数仪测定WBC、Hb和PLT结果一致,但CD1700测定RBC结果的偏倚临床不接受,应采取适当的校正措施。虽然2台仪器均采用原装试剂,检测结果具有溯源性,但为保证其结果的可比性,应进行结果比对和偏倚评估。     

系统化整体护理在婴儿维生素K缺乏迟发型脑出血中的应用

目的通过对婴儿维生素K缺乏致颅内出血的观察及护理,总结此类病例最佳的护理方式。方法对45例婴儿迟发型维生素K缺乏致颅内出血患儿的诱因、出血类型、治疗、预后资料进行性详细地记录,给予系统化整体护理。结果显效33例,有效7例,无效5例。结论精心治疗及系统化整体护理,是患儿恢复健康的保证。维生素K缺乏迟发型颅内出血应预防为主。     

乳腺增生丸的制备及临床观察

乳腺增生症是乳腺结构不良症中常见的一种良性病变。乳腺结构不良症为女性常见病 ,多发病 ,是一类严重影响妇女身心健康、生活及正常工作的疾病。笔者对 2 2 5例乳腺增生症的患者坚持服用我院科研方乳腺增生丸治疗 ,取得较满意的疗效。现报告如下。1　处方与制备1.1　处方元参 2 0 0 g,夏枯草 2 0 0 g,海藻 13 0 g,昆布 13 0 g,云木香 160 g,鸡血藤 2 0 0 g,红花 12 0 g,丹皮 80 g,赤芍 60 g,蒲公英 2 0 0 g,连翘 80 g,花粉 80 g,汗三七 10 0 g,元胡 5 0 g。1.2　制备混合粉碎成细粉 ,过 10 0目筛 ,混合 ,过重筛 ,每 10 0 g药粉加炼蜜 110～…     

右美托咪定辅助硝酸甘油控制性降压在鼻内镜手术中的应用效果

目的：观察右美托咪定辅助硝酸甘油控制性降压在鼻内镜手术中的应用效果。方法2012年4月~2013年2月,选择择期行鼻内镜手术的患者60例,ASAⅠ~Ⅱ级,随机分为硝酸甘油组(N组)和右美托咪定复合硝酸甘油组(DN组),每组30例。记录麻醉前(T0)、手术开始前(T1)、降压15 min(T2)、降压30 min(T3)、和停止降压20 min(T4)各时点的MAP、HR、血乳酸(Lac)浓度及NT值,评估术野质量,并记录苏醒期躁动发生率等。结果与T0时比较,N组MAP于T2、T3时降低(P<0.05),HR于T2~T4时加快(P<0.05),DN组MAP于T1~T3时降低(P<0.05),HR于T1时减慢(P<0.05);与N组比较,DN组MAP及HR于T1~T4时降低(P<0.05)。与T0时比较,N组及DN组血乳酸于T2、T3时升高(P<0.05);DN组术野质量评分低于N组(P<0.05),苏醒期躁动发生率低于N组(P<0.05)。结论右美托咪定辅助硝酸甘油控制性降压,术野理想,为鼻内镜手术一种较适宜的控制性降压方法。     

传染性单核细胞增多症的实验室诊断

<正> 传染性单核细胞增多症又名腺热病,是一种急性的网状内皮系统增生性疾病。病程常具自限性。临床上表现为不规则发热、咽喉炎、淋巴结肿大和脾肿大,血中淋巴细胞增多并有异常淋巴细胞出现,血清中可测得嗜异性凝集素及EB病毒抗体。1.临床资料及实验室检查 患者系1989—1999年间本院门诊及住院病人20例,其中男13例,女7例,年龄均在12—35岁,均有发