阴道毛滴虫AK基因原核表达质粒的构建及在大肠埃希菌中的表达

目的 构建阴道毛滴虫氢化酶体腺苷酸激酶(adenynate kinase,以下简称AK)基因原核表达质粒并予以表达.方法 AK DNA的克隆载体PMD-18T-AK经限制性内切酶BamHI和XbaI双酶切,得到含这2个酶切位点之AKDNA片段,T4连接酶连接到含有2个酶切位点的原核表达载体PUC18,构建出重组原核表达质粒PUC18-AK,经PCR、酶切鉴定.重组质粒PUC18-AK经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,在大肠埃希菌中进行初步表达.结果 AK基因体外扩增产物大小均为690 bp,重组原核表达质粒经PCR及酶切鉴定表明获得正确重组子.在大肠埃希菌中表达出AK蛋白,经十二烷基磺酸钠一聚丙烯酰胺凝胶电泳(SDS-PAGE)分析与理论预测值相符.经蛋白质印迹法(westerm blotting)鉴定具有免疫原性.结论 成功地构建了AK基因原核表达质粒,并在大肠埃希菌中表达了AK蛋白.     

小鼠抵抗素基因及其反义核酸真核表达体系的构建与鉴定

目的构建载有小鼠抵抗素(resistin)基因及其反义核酸的重组真核表达质粒,为下一步进行resistin生物功能研究打基础。方法用resistin基因mRNA编码区序列特异引物,从小鼠脂肪组织中,通过RT-PCR的方法合成resistin cDNA,T4DNA连接酶将resistin cDNA克隆于pGEM-T载体,经双酶切及测序鉴定克隆成功后再亚克隆于pcDNA3.1(+)或pcDNA3.1(-)真核表达载体,并测序鉴定。结果PCR产物长度与resistin cDNA理论长度363bp相符;重组pGEM-T被EcoRⅠ和XbaⅠ内切酶切为约3000bp和355bp两个片段,测序结果表明插入pGEM-T的DNA片段的核苷酸序列与小鼠resistin基因mRNA编码区序列完全一致。重组pcDNA3.1(+)和pcDNA3.1(-)测序结果表明插入的DNA片段分别与小鼠resistin基因mRNA编码区序列和反义resistin基因mRNA编码区核苷酸序列一致。结论成功克隆载有resistin基因和载有resistin基因反义核酸的重组真核表达质粒。     

幽门螺杆菌GroEL基因真核表达载体构建及鉴定

目的构建幽门螺杆菌热休克蛋白GroEL基因的真核表达载体pcDNA3.0-GroEL,为幽门螺杆菌的基因疫苗研制提供参考依据。方法提取幽门螺杆菌基因组DNA,PCR扩增GroEL基因,克隆至pMD19-T载体,通过PCR、酶切及测序鉴定后,将GroEL基因片段用限制性内切酶切下,克隆至真核表达载体pcDNA3.0,构建pcDNA3.0-GroEL重组质粒;采用PCR、酶切对重组质粒pcDNA3.0-GroEL进行鉴定。结果扩增出幽门螺杆菌GroEL基因片段约1 640 bp;pcDNA3.0-GroEL重组质粒经XhoⅠ和EcoRⅠ双酶切,产生1个与GroEL基因PCR产物大小一致的小片段和1个不同于pcDNA3.0-GroEL重组质粒的大片段,表明GroEL基因已成功插入pcDNA3.0质粒中。结论成功构建幽门螺杆菌GroEL基因真核表达载体pcDNA3.0-GroEL。     

幽门螺杆菌Lpp20真核表达重组载体的构建及鉴定

目的以幽门螺杆菌外膜脂蛋白Lpp20为目的基因,构建H.pylori Lpp20基因的真核表达重组载体pcD-NA3.1( )-Lpp20,为H.pylori核酸疫苗的研制奠定实验基础。方法用Pri mer5.0软件分析GenBank中H.pylori外膜脂蛋白Lpp20基因序列,设计合成相应特异性引物,引入BamHⅠ和XhoⅠ酶切位点,以H.pylori26695株基因组为模板,PCR扩增Lpp20目的基因片段,定向插入真核表达载体pcDNA3.1( )多克隆酶切位点中,构建重组体pcDNA3.1( )-Lpp20;通过酶切分析,PCR鉴定及测序鉴定,筛选阳性重组体。结果以H.pylori26695株基因组DNA为模板扩增出特异的Lpp20基因片段,大小约为528bp;双酶切及测序鉴定证明成功构建了H.pylori真核表达重组载体pcD-NA3.1( )-Lpp20。结论PCR扩增得到了大小约为528bp的H.pylori Lpp20目的基因片段;并成功构建了pcD-NA3.1( )-Lpp20真核表达重组载体。     

pcDNA3．1（＋）／NspA真核表达载体的构建及鉴定

目的构建pcDNA3.1( )淋病奈瑟菌外膜蛋白—奈瑟菌表面蛋白A(Neisseria surface Protein A,NspA)基因真核表达载体,为研制淋病奈瑟菌核酸疫苗奠定基础。方法根据淋病奈瑟菌NspA基因序列,设计合成一对引物,用PCR方法从淋病奈瑟菌基因组DNA中扩增出NspA基因片段,将扩增的产物连接于测序载体pUCm-T上,并构建重组体pcDNA3.1( )/NspA,进行酶切、PCR及测序鉴定。结果NspA基因体外扩增产物大小约为525 bp。所构建的pcDNA3.1( )/NspA重组体经双酶切及PCR鉴定,与预期片断的大小相符;测序结果与GenBank中收录的NspA全长序列一致,表明pcDNA3.1( )/NspA真核表达载体构建正确。结论成功地构建了淋病奈瑟菌真核重组表达载体pcD-NA3.1( )/NspA,为NspA蛋白的功能研究和淋病奈瑟菌核酸疫苗的研制提供了物质基础。     

帕金森病PINK1蛋白真核表达载体pcDNA3．1-myc／PINK1的构建及其在COS-7细胞中的表达

目的构建帕金森病PINK1基因真核表达载体pcDNA3．1-mye／PINK1,检测其转染COS-7细胞后的表达。方法用PCR方法从人cDNA文库DNA中扩增PINK1全长cDNA,该基因片段两端设计了EcoR I和BamH I限制性酶切位点。将所得片段连接到T载体上测序证实。利用重组DNA技术将PINK1的cDNA构建到真核表达载体pcDNA3．1-myc—his（-）B上,酶切鉴定。通过脂质体介导的转染技术将所构建的载体导入COS-7细胞中,体外培养,于转染48h后利用RT—PCR和Western Blotting方法检测目的基因的表达情况。主要观察指标：（1）PINK1基因cDNA扩增产物检测。（2）pcDNA3．1-myc／PINK1重组体酶切鉴定。（3）Western—blotting。结果经琼脂糖凝胶电泳及测序证实,PCR获得的片段与GenBank中登记的PINK1序列完全相同;pcDNA3．1-myc／CHIP酶切片段的长度与理论大小相符;转染48h后的COS-7细胞可检测到PINK1蛋白的表达。结论PINK1蛋白真核表达载体构建成功,在COS-7细胞中可获得表达。     

重组人促红细胞生成素四环素调控真核表达载体的构建与鉴定

目的构建并鉴定含有四环素调控的pTRE顺式作用元件的质粒型载体pTRE2hyg-rhEPO。方法设计含BamHⅠ和ClaⅠ酶切位点的hEPO基因引物，采用RT-PCR法从含有rhEPO基因的CHO细胞中克隆hEPO基因片段，亚克隆至pTRE2hyg真核表达载体。转化感受态大肠杆菌Tbp10，酶切鉴定阳性重组子，并进行序列测定。结果经RT-PCR能扩增出604bp的片段，与预期片段大小相符，构建的重组体经酶切鉴定能切出插入片段，测序结果与预期序列完全一致。结论成功构建了重组人EPO四环素调控真核表达载体pTRE2hyg-rhEPO。     

小鼠抵抗素基因及其反义核酸真核表达体系的构建与鉴定

目的构建载有小鼠抵抗素（resistin）基因及其反义核酸的重组真核表达质粒,为下一步进行resistin生物功能研究打基础。方法用resistin基因mRNA编码区序列特异引物,从小鼠脂肪组织中．通过RT—PCR的方法合成resistin cDNA,T4DNA连接酶将resistin cDNA克隆于pGEM-T载体,经双酶切及测序鉴定克隆成功后再亚克隆于pcDNA3．1^（＋）或pcDNA3．1^（-）真核表达载体,并测序鉴定。结果PCR产物长度与resistin cDNA理论长度363 bp相符;重组pGEM—T被EcoR Ⅰ和Xba Ⅰ内切酶切为约3000bp和355bp两个片段,测序结果表明插入pGEM-T的DNA片段的核苷酸序列与小鼠resistin基因mRNA编码区序列完全一致。重组pcDNA3．1^（＋）和pcDNA3．1^（-）测序结果表明插入的DNA片段分别与小鼠resistin基因mRNA编码区序列和反义resistin基因mRNA编码区核苷酸序列一致。结论成功克隆载有resistin基因和载有resistin基因反义核酸的重组真核表达质粒。     

卡氏肺孢子虫MSG抗原基因片段真核表达质粒构建与表达

[目的]构建大鼠源卡氏肺孢子虫MSG抗原基因片段真核表达质粒,进行表达。[方法]以卡氏肺孢子虫DNA为模板,应用PCR扩增MSG基因片段,连接至pGEM-T Easy载体,再克隆至PCDNA3.1( )载体。构建的重组表达质粒经酶切、PCR鉴定后转染至COS-7细胞,并进行大量增殖。RT-PCR验证转染基因的表达。[结果]重组表达质粒PCDNA3.1( )/MSG经酶切及PCR鉴定结果表明构建成功。RT-PCR结果显示MSG基因片段成功转染至COS-7细胞,并在其中进行基因表达。[结论]成功构建了PCDNA3.1( )/MSG重组质粒,并在COS-7细胞中表达,为进一步进行核酸疫苗的研究打下基础。     

长春新碱过量引起严重毒副反应1例的护理体会

报道1例霍奇金淋巴瘤患者因误用长春新碱（VCR）10mg一次性静脉推注后治疗护理情况。其出现间断性神志恍惚、眼睑闭合不全、言语不清、口腔黏膜糜烂、全身疼痛、麻痹性肠梗阻、尿潴留、手足麻木等症状,经积极解救,禁食,持续胃肠减压、胃管内注入麻油、开塞露、生理盐水灌肠,合理应用肠外营养,注重疼痛、心理护理,做好口腔、肛周护理,预防感染加重,患者病情得到控制好转出院。     

Choice of measures of vaccination and estimates of risk of pediatric pertussis

BackgroundVaccination uptake at the individual level can be assessed in a variety of ways, including traditional measures of being up-to-date (UTD), measures of UTD that consider dose timing, like age-appropriate vaccination, and risk reduction from individual doses. This analysis compared methods of operationalizing vaccination uptake and corresponding risk of pertussis infection.MethodsCity-wide case-control study of children in Philadelphia aged 3 months through 6 years, between 2001 and 2013. Multiple logistic regression was used to isolate the independent effects of each measure of vaccination uptake and the corresponding relative odds of pertussis.ResultsBeing UTD on vaccinations was associated with a 52% reduction in risk of pertussis (OR 0.48, 95% CI: 0.34, 0.69). Evaluation of delayed receipt of vaccine versus on-time UTD yielded similar results. There was a decrease in risk of pertussis for each additional dose received with the greatest reduction in pertussis infection observed from the first (OR 0.48, 95% CI: 0.28, 0.83) and second dose (OR 0.17, 95% CI: 0.08, 0.34). Additional doses conferred minimal additional protection in this age group.ConclusionExamining vaccination status by individual doses may offer improved predictive capacity for identifying children at risk for pertussis infection compared to the traditional UTD measure.     

上海市某医院骨科平均住院日影响因素分析

对上海市某医院2003年-2007年骨科出院病人的住院日描述性分析．2003年-2007年骨科的床位利用指数与平均住院日相关性分析．2003年-2007年骨科床位与医护比例分析．2007年骨科前10大病种平均住院目影响因素分别进行单因素相关性分析和多因素逐步回归分析（STATA软件）。通过对骨科10大病种住院日影响因素分析,术前等待天数、手术类型、是否输血分别对10个、9个和8个病种的住院目有影响。输血因素和手术类型是医院不可控、由病人的病情决定的,术前等待天数是管理因素,是最值得医院重视的影响因素。     

尘肺病患者145例死因调查

目的分析汉中市尘肺病的发生、发展和死亡原因，以提高尘肺病患者的生存质量。方法对145例尘肺病患者的死因进行回顾性分析，计算其平均发病年龄、死亡年龄及患尘肺病后的存活年限。结果矽肺的平均发病年龄和死亡年龄均早于煤工尘肺和石棉肺，尘肺病的主要死因为患肺部疾患，患尘肺病后的平均存活年限为12a。结论加强职业健康体检工作，对尘肺病人做到早发现早治疗和预防并发症的发生。     

Correlates of stages of change of smoking among inhabitants of deprived neighbourhoods

BACKGROUND: This study examines the prevalence and correlates of stages of change of smoking, in terms of psychosocial, structural and sociodemographic factors, among inhabitants of deprived neighbourhoods. METHODS: Cross-sectional data were obtained from a survey on health related behaviour. Subjects were 2009 current and former smokers, aged 20-46, living in deprived neighbourhoods in Rotterdam, the second largest city in the Netherlands. Three groups of smokers were formed according to the stages of change-definitions of the Transtheoretical Model: smokers not planning to quit (precontemplators), smokers planning to quit (contemplators/preparators) and former smokers (actors/maintainers). Smokers planning to quit and smokers not planning to quit were compared regarding psychosocial factors (attitude, social norm, self-efficacy), structural factors (neighbourhood problems, material deprivation, financial problems, employment status) and sociodemographic factors (age, gender, marital status, cultural background, educational level). Former smokers were compared with smokers planning to quit regarding structural and sociodemographic factors. Logistic regression was used to assess correlates of stages of change. RESULTS: Smokers planning to quit (prevalence = 19%) reported a more positive attitude, stronger social norms and higher self-efficacy expectations in quitting smoking than smokers not planning to quit (prevalence = 57%). Smokers planning to quit less often were Dutch-born, more often had attended higher vocational schooling or university and more often reported experiencing two or more neighbourhood problems compared to smokers not planning to quit. Former smokers (prevalence = 24%) were older, more often Dutch-born, married, employed and higher educated, compared to smokers planning to quit. Furthermore, former smokers less often reported material deprivation and financial problems than smokers planning to quit. CONCLUSION: Among people living in deprived neighbourhoods, different factors correlate with different stages of change of smoking. Implications for health promotion are discussed.     

西林瓶装注射用水的装量检查分析

为了检查西林瓶装注射用水的实际分装量是否符合2005年版《药典》三部要求,按照2005年版《药典》三部中“生物制品分装和冻干规程”,分装规格为0．8ml的西林瓶装注射用水10000瓶,随机在分装过程的前、中、后阶段共抽取100瓶,轧盖、目检。分别用1ml标定注射器及1ml普通无菌注射器抽取每瓶注射用水的实际体积,以t检验法对实际抽取量与标示量的差值进行统计学分析,结果显示其与2005年版《药典》三部要求分装附加量为0．1ml相比有显著性差异（P〈0．001）。     

对《医疗事故处理条例》的几点认识--学习《医疗事故处理条例》的一点体会

国务院颁布的《医疗事故处理条例》（以下简称《条例》）在吸收、总结原《医疗事故处理办法》（以下简称《办法》）实施十几年以来正反两方面经验教训的基础上，对《办法》作了许多重大的修改：《条例》扩大了医疗事故的主体与范围，提出了防范医疗事故的一些重要措施，授权医学会独立担纲医疗事故技术鉴定工作，赋予了患者更多的权利，规定医疗事故民事责任争议可以“私了”，制定了更为规范的医疗事故的赔偿制度等。《条例》作出的这些新规定必将使医疗事故得以更加科学、客观、公正、公平、合理的解决。认真学习、把握《条例》的这些新规定，很好地透视《条例》的立法宗旨与原则，必将极大地提高学习、宣传《条例》的主动性与积极性，更好地推进《条例》的贯彻执行工作。     

The analysis of case-control studies of the efficacy of screening for recurrence of cancer

Occasionally, the efficacy of screening for cancer recurrence has been evaluated by means of case-control studies. However, these studies generally have not been designed and analyzed in a manner that will yield an unbiased result. The commentary shows that by adhering to the principles developed for the analysis of studies of the efficacy of screening for the presence of a given cancer in persons with no history of this disease, a case-control study of screening for recurrence will improve its chances of obtaining valid findings.     

137例地中海贫血出生相关因素分析

目的:揭示地中海贫血患儿出生相关因素,提出干预措施,为进一步制定地贫预防控制总体规划提供决策参考。方法:对近3年137例地贫儿(45例重型β-地贫、29例中间型β-地贫、32例中间型α-地贫和31例巴氏胎儿水肿综合征)出生相关因素进行调查研究。结果:地贫儿出生相关社会心理因素顺位依次为:基因携带者不知情(70.07%)、经济困难(21.17%)、心理因素(5.11%)、其它(3.65%)。基因携带者对自身情况不知情是本组地贫儿主要出生相关因素,与其它出生相关因素有显著差异(P<0.05)。结论:基因携带者自身的认识与重视是预防控制工作的一个重要组成部分,地贫高发区人群携带者(轻型β-地贫、α-地贫特征和静止型携带者)的检出及系统管理是预防控制工作的关键环节,应进一步应用生物、社会及心理的医学模式搞好地贫预防控制工作。     

可乐对小鼠精子质量影响

目的 探讨可乐对雄性小鼠精子质量及数量的影响.方法 31只昆明种雄性小鼠按体重随机分为阴性对照组、低剂量组、高剂量组(分别以0.6ml生理盐水、0.3,0.6ml可乐灌胃),腹腔注射组(0.6ml可乐注射)和阳性对照组(0.6ml环磷酰胺腹腔注射)5组.灌胃及腹腔注射1次/d,连续7d,测定小鼠精子的运动参数,并观察其畸形发生率.结果 高剂量组肾脏系数与阴性对照、低剂量组比较差异均有统计学意义(P<0.05);腹腔注射组与阴性对照组比较小鼠附睾系数明显升高(P<0.05).腹腔注射组的精子平均移动角度与阴性对照组比较明显升高(P<0.05),低剂量组精子密度与阳性对照组比较差异有统计学意义(P<0.05).高剂量组、阳性对照组与阴性对照组比较精子畸形率明显升高(P<0.05);低、高剂量组及腹腔注射组与阳性对照组比较差异均有统计学意义(P<0.05);低、高剂量组与腹腔注射组比较差异均有统计学意义(P<0.05).结论 可乐对小鼠精子活性和形态有一定的影响.