人巨细胞病毒gB基因的构建、表达及鉴定

目的构建人巨细胞病毒（HCMV）gB基因,并对pET28a／gB重组质粒进行诱导表达,鉴定表达的目的蛋白。方法根据Genebank中HCMV核苷酸序列设计引物,并在引物的5’、3’端分剐加入BarnHI、EcoRI限制性内切酶位点。特异性扩增gB编码基因片段,同时构建舍HCMVgB编码基因的原核表达载体,对重组质粒进行诱导表达,用间接酶联免疫吸附测定（ELISA）法对纯化的Ni／gB融合蛋白的灵敏度和特异度等生物活性进行鉴定,灵敏度可达95％,特异度达96．7％。结果目的蛋白经诱导后表达于上清液中,经鉴定,目的蛋白保留了天然蛋白原有的生物活性。结论Ni／gB融合蛋白的获得,为研制以重组蛋白代替传统全病毒作为检测抗原的新型HCMV特异性免疫学检测试剂盒奠定了基础。     

EGFL7原核表达载体的构建及鉴定

【目的】构建类表皮生长因子域7（EGFL7）原核表达载体,并诱导其表达、纯化及鉴定目的蛋白。【方法】以人肝癌细胞系 HepG2总RNA为模板,PCR扩增 EGFL7基因,克隆至原核表达载体pET‐21a（＋）中,转化大肠杆菌 BL21（DE3）,IPTG 诱导表达。 His‐tag 磁珠纯化重组蛋白 EGFL7,SDS‐PAGE 鉴定。【结果】克隆目的基因序列正确,未发生碱基突变;重组表达质粒pET21a（＋）‐TRX‐EGFL7经双酶切鉴定构建正确;表达的重组蛋白相对分子量为80 kD ,与预期相符。【结论】成功在大肠杆菌BL21（DE3）中表达并纯化了EGFL7重组蛋白,为后续研究奠定了基础。     

小鼠A细胞介素21原核表达载体的构建及活性鉴定

目的：白细胞介素21是近年发现的由T细胞分泌的细胞因子，具有广泛的免疫调节作用和抗肿瘤活性。为进一步观察其体内外免疫作用，拟构建小鼠白细胞介素21（mlL-21）原核表达载体，并对其表达的活性进行鉴定。 方法：实验于2006—03／2007—05在南京医科大学附属南京儿童医院儿科研究所和东南大学基础医学院病原生物学与免疫学系完成。实验材料：实验中所用BALB／c小鼠由扬州大学比较医学中心提供，六七周龄，雌雄兼备，实验过程中对动物处置符合动物伦理学标准。pcDNA3．1／mlL-21质粒由东南大学基础医学院免疫学实验室提供。实验方法：以pcDNA3．1／mlL-21质粒为模板，PCR获得长度为369bp的mlL-21基因片段。将其定向克隆至pET-28a（＋）质粒中，采用PCR酶切和测序等方法对重组质粒进行鉴定。并对重组蛋白进行IPTG诱导表达、Western blot及体外生物学活性检测。 结果：构建了重组原核表达载体pET-28a（＋）／mlL-21，经鉴定后证实重组质粒构建正确。SDS-PAGE显示含重组质粒的诱导菌在M16000位置出现一蛋白条带。Western blot结果显示该蛋白具有良好的免疫原性。体外生物学功能检测显示该重组蛋白能够增强小鼠脾细胞中的NK细胞杀伤活性。 结论：成功构建了重组原核表达载体pET-28a（＋）／mlL-21，表达的mlL-21具有良好的免疫原性。     

人EGFL7基因真核表达载体构建及鉴定

【目的】构建人类表皮生长因子域7（EGFL7）基因的真核表达载体。【方法】通过聚合酶链式反应（PCR）方法扩增EGFL7目的片段,利用基因重组技术构建pEGFPC1-EGFL7真核表达载体,并进行酶切和测序鉴定。鉴定正确的克隆瞬时转染293T细胞,通过Westernblot方法检测目的蛋白的表达。【结果】通过酶切和测序鉴定,pEGFBC1-EGFL7真核表达载体序列正确,编码框正确。转染后的293T细胞经Westernblot检测,能够表达目的蛋白。【结论】本方法成功构建了EGFL7真核过表达载体,为进一步研究其功能奠定了基础。     

C反应蛋白质粒的构建表达及纯化

目的 在大肠埃希菌中表达和纯化C反应蛋白（CRP），并鉴定纯化蛋白的免疫反应性，为CRP的检测打基础。方法 应用逆转录（RT）PCR方法，从人肝细胞文库中扩增人C反应蛋白cDNA基因片段，与表达质粒载体pCRT7／NTTOPO重组，获得表达质粒CRP—pCRT7／NT。用氨苄青霉素平板筛选转化子，双酶切与DNA测序鉴定CRP基因表达质粒pCRT7／NTTOPO载体的整合状态，用IPTG诱导表达，并经组氨酸亲和层析柱纯化获得CRP的蛋白纯品。采用Western印迹的方法鉴定纯化蛋白的免疫反应性。结果 利用表达载体pCRT7／NTTOPO载体表达的含6个组氨酸标签的CRP，在SDS—PAGE上出现1条相对分子质量约30000的阳性条带，经组氨酸亲和层析柱纯化后用Western印迹杂交证实为目的蛋白。结论 成功地构建了在大肠埃希菌表达CRP的质粒，建立了蛋白纯化的系统，为下一步CRP检测试剂盒的制备打下了基础。     

构建人 COX-2反义核酸真核表达载体对膀胱癌 T24细胞株COX-2表达的作用

【目的】构建人环氧化酶-2（COX-2）反义核酸真核表达载体,探讨其抑制膀胱癌 T24细胞COX-2表达的作用。【方法】经PCR法从含人COX-2基因的质粒中扩增其cDNA ,通过限制性内切酶克隆于 T载体EcoR Ⅰ和Xba Ⅰ位点之间,并将其反向插入pcDNA3．1中获得重组质粒。应用脂质体介导将其导入T24膀胱癌细胞中,经过G418筛后进行流式细胞术、间接免疫荧光染色和RT-PCR鉴定。【结果】核酸测序和限制性内切酶消化证实重组COX-2反义核酸真核表达质粒 pcDNA3．1/COX-2 as是正确的,经鉴定转导 pcD-NA3/COX-2 as的T24细胞中环氧化酶-2的表达明显抑制。【结论】成功构建人COX-2反义核酸真核表达载体并获得环氧化酶-2基因表达持续下调的膀胱癌细胞株T24-AS。     

ICAM-1-GFP的构建及其与Molt-4细胞的结合

本研究构建人细胞间黏附分子1（ICAM-1）全长基因的真核表达载体pEGFP—CI—ICAM—1,转染中国仓鼠卵巢细胞（CHO—K1）细胞株,并检测其在cH0细胞中的表达及与Molt-4细胞的结合。采用RT—PCR法从健康人外周血中分离单个核细胞,钓取ICAM-1全长基因（1622bp）,与pMD18-T载体连接做全自动序列测定。将测序正确的克隆质粒pMD18-T—ICAM-1和表达载体pEGFP—C1分别用HindⅢ和Ⅰ进行双酶切,应用基因重组技术构建ICAM-1全长基因真核表达载体pEGFP—C1-ICAM-1,质粒经Hindm和SacⅡ双酶切和PCR电泳鉴定后,采用脂质体转染法转染CHO细胞,并进行G418筛选。用RT—PCR、流式细胞术和荧光显微术检测ICAM-1-GFP的表达及亚细胞的定位,用检测ICAM-1-GFP／CHO细胞与Molt-4细胞的结合能力评价ICAM．1-GFP融合蛋白的功能。结果表明：重组质粒经限制性酶切鉴定得到与ICAM—1全长基因长度-致（1622bp）的酶切产物;测序分析证实,PCR产物与GenBank上登录的ICAM-1基因（NM-000201）序列完全-致,表明成功地完成了ICAM-1的扩增和表达载体的构建;荧光显微镜下可见转染的CHO细胞有绿色荧光蛋白的表达,表达的融合蛋白较均匀地分布于整个细胞：FACS检测ICAM．1-GFP的荧光转染率为（13±5．5）％,表明ICAM—1—GFP基因进入到CHO细胞并获得了有效表达,成功构建了ICAM-1-GFP／CHO细胞,并且ICAM-1-GFP／CHO细胞能够结合PMA处理的Molt-4细胞。结论：成功构建了ICAM-1-GFP真核表达载体,构建的ICAM—1—GFP真核表达载体在CHO细胞内稳定表达,／CAM—1—GFP／CHO细胞能与M0lt-4细胞结合,这为进-步研究ICAM-1分子的功能打下基础。     

pTracer-CMV／Bsd-BDNF真核表达载体的构建及表达

【目的】构建人脑源性神经营养因子（BDNF）基因真核表达载体,并在小鼠耳蜗毛细胞与螺旋神经节细胞（SGCs）表达。【方法】应用RT‐PCR从人外周血中扩增BDNF基因开放阅读框（ORF）,采用 TA克隆技术,将目的片段插入到pUCm‐T载体中进行鉴定,重组的质粒命名为pUCm‐T‐BDNF。随后,将BDNF进一步克隆到真核表达载体pT racer‐CM V／Bsd中。用脂质体将经过测序、验证的重组pT racer‐CM V／Bsd‐BD‐NF质粒转染小鼠耳蜗SGCs细胞,荧光显微镜下观察绿色荧光蛋白的表达,转染SGCs细胞,经杀稻瘟菌素（Blasticidin）筛选4周,用Western blot检测BDNF的表达。【结果】成功构建了pTracer‐CMV／Bsd‐BDNF真核表达质粒,转染SGCs细胞,发现转染成功的细胞均发绿色荧光;Western blot检测结果表明,所转染的BD‐NF在SGCs细胞中成功表达。【结论】成功构建了能够同时表达绿色荧光蛋白和BDNF的真核表达质粒pTracer‐CMV／Bsd‐BDNF ,为进一步研究BDNF在内耳基因治疗中的作用奠定了基础。     

丙型肝炎病毒E1基因在COS-7中的表达

目的：了解丙型肝炎病毒（HCV）E1基因在真核细胞中的表达情况。方法：采用基因重组技术，构建含HCVE1基因的重组表达质粒pEE1/HisC-E1，经酶切和PCR鉴定后，用脂质体转染法将重组质粒导入COS-7进行表达，经蛋白印迹实验鉴定表达产物。结果：重组表达载体pEF1/HisC-E1在COS-7中获得表达，表达的E1糖蛋白约29kD，具有抗原性，结论：HCVE1基因在真核细胞高效表达，为进一步研究HCVE1糖蛋白的生物学特性奠定基础。     

人骨形成蛋白2真核表达载体的构建和体外表达

背景：骨形态发生蛋白2（bone morphogenetic protein2，BMP-2）是已知的所有生长因子中对骨的形成作用最强的生长因子，被认为是最具有前途的骨诱导物质。目的：构建人骨形成蛋白2真核表达载体并观察其体外表达情况。设计、时间及地点：自身对照实验，于2005-07／2006-05在华中科技大学同济医学院分子生物中心实验室完成。材料：pcDNA3．1（＋）载体由华中科技大学同济医学院左石博士惠赠；成骨肉瘤组织由华中科技大学同济医学院附属协和医院骨科提供。方法：从人成骨肉瘤细胞中提取细胞总RNA，利用反转录．聚合酶链反应方法扩增获得人BMP-2基因cDNA，将基因片断重组到pGEM-T质粒中构建pGEM．T-hBMP-2重组质粒载体，转化到大肠杆菌DH5n后筛选阳性克隆，利用限制性酶切和DNA序列分析鉴定重组质粒。分别用RcoRI和NotI双酶切pGEM-T-hBMP-2质粒和pcDNA3．1真核表达载体，将克隆载体中人骨形成蛋白2基因重组到pcDNA3．1真核表达载体，提取质粒作酶切电泳、聚合酶链反应鉴定及DNA测序后，用脂质体体外转染小鼠骨髓基质细胞，反转录-聚合酶链反应检测BMP．2的表达。主要观察指标：①人骨肉瘤细胞总RNA反转录-聚合酶链反应结果。②重组质粒pGEM．T-hBMP-2和pcDNA3．1-hBMP-2的构建和酶切鉴定。③BMP-2在小鼠骨髓基质细胞内的表达。结果：人骨肉瘤细胞总RNA经反转录-聚合酶链反应扩增后，获得1．2kb条带。经酶切电泳、聚合酶链反应鉴定及DNA测序证实实验成功克隆BMP-2基因，重组质粒pcDNA3．1-hBMP-2构建正确；该重组质粒能在体外培养的小鼠骨髓基质细胞中有效表达BMP-2。 结论：实验成功克隆人骨形成蛋白2基因并构建了此基因的真核表达载体。     

现代残疾康复理念、政策与社区康复体系研究

本研究分析了当代残疾康复的理念和理论架构,构建了以国际公约、决议和政策、国内法律和政策以及操作性工具3个层次的康复理论体系;运用包容性发展,分析探讨了当代社区康复的体系和特点;就发展康复事业、为残疾人提供全面系统的康复服务提出相关的政策建议。     

国内外唇腭裂专科护理发展现状的文献研究及分析

目的了解和分析国内唇腭裂专科护理工作发展的现状。方法检索并分析万方医学网、中国知网、Springer Link及PubMed英文数据库近6年有关唇腭裂手术的围术期护理、患儿的喂养、手术安全管理、围术期疼痛研究以及心理护理的相关文献。结果共检索出国内文献89篇(核心期刊20篇)、国外文献22篇,其中围术期护理38篇、患儿的喂养16篇、手术安全管理10篇、围术期疼痛研究10篇、心理护理15篇。结论与国外文献相比,国内论文总结和回顾性居多,应增加研究探讨性论文,从而促进唇腭裂临床护理工作专科化的发展。     

城乡学童个性行为特征调查

目的探讨农村与城市儿童的个性行为特征。方法将97名农村学龄儿童设为农村组,97名城市学龄儿童设为城市组,采用艾森克个性问卷一儿童版和Achenbach’s 行为量表一儿童版进行评定分析。结果两组学龄儿童艾森克个性问卷各维度评分均无显著性差异（P均〉0．05）,且与全国常模相一致（P均〉0．05）;农村组男学童Achenbach’s行为量表违纪行为、攻击行为及外向型因子分均显著高于城市组,女学童退缩、社交问题因子分均显著高于城市组（P〈0．05或0．01）,其他因子分均无显著性差异（P均〉0．05）。结论农村与城市儿童个性无显著差异,而农村儿童存在较多的行为问题,可能与其所受教养和生活方式有关。     

我院文职护士管理和培训的实践与体会

目的构建文职护士培训、使用及管理的科学方法。方法通过规范管理、科学施训、作为骨干大胆任用等多种管理方式,使文职护士得到充分的发展。结果文职护士群体成长为医院优秀骨干,首批满3年的文职护士顺利通过续聘考核。结论科学的管理培训是文职护士成长的重要基石,对医院护理管理有着重要作用。     

Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania, Trypanosoma, and Plasmodium

Molecular tools continue to be important in the prevention and control of parasitic diseases. However, using these techniques directly in the field remains a major challenge. Therefore, the preservation of clinical samples collected from endemic field areas for later analysis remains an important preanalytical process. This study aimed at identifying a suitable protocol for stabilization and preservation of RNA and DNA in bioclinical specimens for Trypanosoma, Leishmania, and Plasmodium research. Both spiked and unspiked blood samples were preserved in 7 protocols (different media; storage temperatures). Samples were evaluated for possible degradation of DNA and RNA along the storage duration up to the 10th week. Nucleic acid targets were assessed as follows: (i) Trypanosoma and Plasmodium RNA analysis was done using real-time nucleic acid sequence-based amplification (RT-NASBA) for 18S rRNA and for stage-specific Pfs25 mRNA, respectively; (ii) Trypanosoma DNA assessment analysis was conducted by using a conventional PCR for 18S rDNA; (iii) Leishmania RNA analysis was performed with a quantitative NASBA for 18S rRNA and Leishmania DNA assessment with an RT-PCR for 18S rDNA. Findings suggested that a newly developed L3™ buffer proved to be reliable and suitable for both short- and long-term preservation of parasite nucleic acid material. This buffer is envisaged to be suitable for utilization in field situations where resources are limited.     

History of violence and subjective health of mother and child

Objective: To study the self-reported prevalence of experienced violence among a cohort of women about two years after giving birth, their health during pregnancy, pregnancy outcomes and their experience of their child’s health.

Setting and subjects: In 2011, a total of 657 women participated in phase III of the Childbirth and Health Cohort Study in Icelandic Primary Health Care, 18 to 24 months after delivery. The women had previously participated in phase I around pregnancy week 16 and phase II 5–6 months after delivery. Data were collected by postal questionnaires.

Main outcome measures: Women’s reported history of experienced violence, sociodemographic and obstetric background, self-perceived health, the use of medications and their child’s perceived health.

Results: In phase III, 16% of women reported experiencing violence. These women felt less support from their current partner (p?p?p?p?p?p?=?0.008).

Conclusions: Our study confirms that a history of violence is common among women. A history of violence is associated with various maternal health problems during and after pregnancy, a higher rate of caesarean sections and maternal reports of health problems in their child 18–24 months after birth.

• KEY POINTS

• Violence is a major concern worldwide. Understanding the impact of violence on human health and developing effective preventive measures are important elements of any public health agenda.

• ??The reported prevalence of experiencing violence was 16% among women attending antenatal care in the primary health care setting in Iceland.

• ??Women with a history of violence reported worse health in general during pregnancy and delivered more often by caesarean section, compared to women with no such history.

• ??Mothers with a history of violence also evaluated the general health of their child as worse than women with no such history.

• ??The findings of this study support the importance of recognizing and addressing experienced violence among women in primary care.

     

Rates and risks of transmission of smallpox and mechanisms of prevention

In 1980, the World Health Organization declared smallpox eradicated from the world; the last known natural case had occurred in Somalia in 1977, and the United States had stopped routinely vaccinating its citizens in 1972. However, with increasing concerns regarding domestic and international terrorism, smallpox has resurfaced as a potential threat to global health. We review the direct and indirect modes of smallpox transmission and how patterns of transmission vary substantially, depending on the severity of circulating disease, vaccination status, environmental and socioeconomic factors, and the setting of an outbreak. We examine mechanisms for controlling outbreaks of disease and preventing further transmission in the event of an outbreak, with an emphasis on smallpox vaccination.     

通用设计原则与产品研发策略

本文介绍了通用设计的概念和设计原则,并阐述了根据此原则实施通用设计的产品研发策略。通用设计惠及的不仅是残疾人,也惠及了包括普通公众在内的广泛人群,以及企业。应将通用设计的原理应融入政府相关政策和未来发展的规划中,为残疾人以及更多人群提供合理便利的通用设计产品。     

Prevalence and severity of nausea and vomiting of pregnancy and effect of vitamin supplementation.

OBJECTIVE: Although nausea and vomiting of pregnancy is the most common medical condition during pregnancy, there are many unanswered questions regarding its cause, epidemiologic features and optimal management. The objectives of this study were to ascertain the prevalence of nausea and vomiting in a sample of Canadian women, to characterize the distribution of their severity and to investigate the role of vitamin B6 deficiency in their etiology. DESIGN: Prospective study. SETTING: Antenatal counselling service for pregnant women. PATIENTS: Three cohorts of women: a prospective, population-based cohort of 193 women, to estimate the rate and severity of nausea and vomiting (cohort A); a cohort of 555 women who sought advice for nausea with or without vomiting, to study the correlation between the maximal daily number of episodes of vomiting and maximal weight loss (cohort B); and a prospective cohort of 301 women who reported vomiting, to correlate vitamin supplementation with vomiting (cohort C). INTERVENTIONS: All 3 cohorts were interviewed during the counselling session, and cohort B was followed up prospectively. OUTCOME MEASURES: Frequency of nausea and vomiting, weight loss, maximal number of daily episodes of vomiting, rate of multivitamin supplementation. RESULTS: Overall, 67% of the women in cohort A reported experiencing nausea or vomiting, or both; 22% reported vomiting, and 9% experienced weight loss. In cohort B there was a significant correlation between the maximal number of daily episodes of vomiting and maximal weight loss, although there was wide variation (r2 = 0.25, p < 0.001). There was a highly significant correlation between the number of daily vomiting episodes and mean weight loss (r2 = 0.99). In cohort C, vomiting was significantly associated with lack of supplementation with multivitamins before 6 weeks' gestation (p = 0.002). CONCLUSIONS: The relation between number of daily vomiting episodes and mean weight loss may serve as a clinical tool to assess the severity of nausea and vomiting in pregnancy and the success of anti-emetics and rehydration regimens. Further study is needed to elucidate the biologic basis of the observed association between vomiting and lack of multivitamin supplementation in early pregnancy.     

胃癌DNA含量与E-cad、EGFR表达的关系及其意义

目的　探讨胃癌组织DNA含量与E cad、EGFR表达的关系 ,评价其在浸润、转移潜能及预后判断中的作用。方法　应用图像分析仪及SP免疫组化法检测 30例正常胃黏膜上皮、30例不典型增生和 5 0例胃癌组织DNA含量与E cad、EGFR蛋白表达情况。结果　①正常胃黏膜上皮、Ⅰ、Ⅱ、Ⅲ级不典型增生及胃癌组的DI、PI、S %、>5c %逐渐增高 ,与胃癌分期、淋巴结转移显著相关 (P <0 0 1)。②E cad在正常胃黏膜上皮阳性表达率 10 0 % ,EGFR呈阴性表达 ;Ⅰ、Ⅱ、Ⅲ级不典型增生及胃癌E cad阳性表达率逐渐降低 ,EGFR的阳性表达率逐渐升高 ,且差异显著 (P <0 0 5 ) ;E cad表达减弱、EGFR表达升高与胃癌分化差、淋巴结转移显著相关 (P <0 0 1)。③E cad表达阴性的胃癌DNA指数及EGFR表达水平显著高于E cad阳性 (P <0 0 1)。结论　检测DNA含量及E cad、EGFR蛋白表达有助于胃癌的早期诊断及转移潜能和预后的判断。