欧猬迭宫绦虫全长cDNA文库构建及鉴定

应用SMART方法成功构建欧猬迭宫绦虫成虫全长cDNA文库,文库的重组率为95.5%,库容量为1.06×106;平均插入片段长度约为1.4kb。选取48个随机阳性克隆测序,去除450bp序列后获得36个有效表达序列标签(EST),平均长度为674bp,其中单基因簇(unigene)比例为58.3%(21/36);26条具有同源性序列的EST中15条有或可能有5′末端,全长性比率为57.7%(15/26)。文库质量良好,可用于大规模EST测序。     

日本血吸虫尾蚴cDNA文库的构建及分析

目的　构建日本血吸虫尾蚴 c DNA文库。　方法　从日本血吸虫尾蚴中提取总 RNA,运用“SMART c D-NA文库构建试剂盒”构建文库。　结果　所建文库的初始滴度为 1.8× 10 7pfu/ ml,经 1次扩增后的滴度为 2 .5× 10 7pfu/ ml,插入子的平均长度为 1.0 75 kb,重组率为 94 .4 % ,并从该文库中调出了日本血吸虫保守基因 TPI和 JF2的全长 c DNA片段。　结论　构建了日本血吸虫尾蚴 c DNA文库     

8～10周龄胎儿心脏cDNA文库大规模表达序列标签分析

目的:研究心脏发育过程中基因表达特点.方法:随机选择8～10周龄胎儿心脏互补脱氧核糖核酸(cDNA)文库的克隆进行表达序列标签(EST)测序,结果与基因库/欧洲分子生物学实验室/日本脱氧核糖核酸数据库(GenBank/EMBL/DDBJ)进行同源性比较,所获EST进行功能分类,并比较其与10～12周龄胎儿及成人心脏表达基因功能分类的不同.结果:2 338条EST中,1 614条69.03%与已知基因匹配,547条(23.4%)与其它EST或基因组DNA相似,177条(7.57%)是新EST.发现了不同心脏发育时期中基因表达的特点.结论:大规模EST分析是系统研究心血管系统不同发育时期基因表达特点的有力工具.     

弓形虫昆山分离株速殖子期表达型cDNA文库的构建及鉴定

目的　构建弓形虫速殖子期表达的基因的完整长度cDNA文库。方法　用CF - 11纤维素柱快速提纯速殖子 ,以盐酸异硫氰酸胍 (AGPC)一步法抽提总RNA ,oligo -dT纤维素分离mRNA后 ,用ClonTech公司的SmartTMPCRcDNA文库构建试剂盒 ,构建了弓形虫昆山分离株的表达型文库。结果　获得 5× 10 6个独立克隆 ,重组率为 99% ,插入片段的平均长度为 1kb。结论　本文库可望用于弓形虫疫苗研究的候选基因的筛选     

PDX-1基因克隆载体的体外构建

目的体外构建胰-十二指肠同源框-1（PDX-1）基因的克隆载体。方法采用TRIzol方法从大鼠胰岛细胞瘤细胞株中提取RNA,通过RT-PCR方法在体外扩增大鼠PDX-1 c DNA,琼脂糖凝胶电泳鉴定,割胶纯化后回收目的基因,与p MD-18T克隆载体连接构建,经限制性内切酶双酶切及DNA序列分析鉴定p MD-18T-PDX-1的正确构建。结果正确构建了含有PDX-1 c DNA的克隆载体p MD-18T-PDX-1,其中PDX-1 c DNA全长为908 bp。将经双酶切鉴定正确的p MD-18T-PDX-1细菌克隆进行测序,测定结果与Gen Bank中大鼠PDX-1基因序列（NM₀22852.3）一致。结论成功构建了PDX-1基因克隆载体。该载体可为PDX-1基因分子生物学研究及PDX-1基因在诱导非β细胞向胰岛素分泌细胞分化中作用机制的研究提供基础。     

细粒棘球蚴全长cDNA文库的构建及初步鉴定

目的　分离目的基因构建cDNA文库的经典方法繁琐且不易得到全长cDNA(3’和 5’端 ,尤其是基因的 5’非翻译区 )。本研究通过构建细粒棘球蚴全长cDNA文库 ,筛选全长目的基因 ,研究其结构和功能。方法　采用TRIZOL试剂提取E .granulosus总RNA ,用ClonTech公司的SMARTTMcDNA文库构建试剂盒构建E .granulosus原头节全长cDNA文库 ,文库的包装使用EICENTERTECHNOLOGIES公司提供的包装蛋白。结果与结论　成功构建细粒棘球蚴全长cDNA文库 ,文库滴度为 1.4× 10 7pfu/ml。文库的重组效率达到 99%以上 ,插入片段长度约为 1.1kb。     

寄生虫cDNA文库的研究

c DNA文库是将细胞中所有的 m RNA逆转录成与之互补的 c DNA,进而合成双链 c DNA,再克隆到载体上 ,引入适当的宿主细胞内繁殖而得。与基因文库相比 ,它有着库小、易筛选且更能直接反映细胞特征的优点。因此 ,c DNA文库得到了广泛的应用 [1 ]。在寄生虫学领域 ,由于克隆和表达寄生虫特异抗原基因的重组 DNA技术的发展 ,构建 c DNA文库作为从分子水平提供虫源抗原的手段 ,已在保护性和诊断性抗原的获取及研究方面起了重要作用。同时 ,c DNA文库有自身的特殊用途 ,即可对寄生虫发育过程中时间调节基因的表达特征 ,以及组织特异性基因…     

粉尘螨全长cDNA文库的构建及初步鉴定

目的构建粉尘螨全长cDNA文库,为粉尘螨相关基因研究奠定基础。方法用RNAiso Reagent试剂盒提取粉尘螨总RNA,然后以SMART IVTM Oligo-nucleotide和CDSIII/3’PCR Primer为引物,在反转录酶作用下合成cDNA第1链,利用LD-PCR方法合成cDNA第2链。合成的双链cDNA经蛋白酶K消化及SfiⅠ酶切,得到的cDNA克隆入λTripIEx2载体中,构建粉尘螨全长cDNA文库,测定分析文库滴度及插入片段大小。结果成功构建粉尘螨全长cDNA文库。检测cDNA文库未扩增时滴度为7.3×106pfu/ml,文库扩增后滴度为5.0×109pfu/ml,插入片段为0.5~2.5kb,平均1.2kb左右。结论成功构建了高质量的粉尘螨全长cDNA表达文库,所建文库容量及插入片段大小适用于对粉尘螨相关基因的进一步研究。     

CHO细胞MAR片段的克隆及其逆转录载体的构建

目的构建重组人促红素（CHO）细胞核基质结合区MAR的逆转录载体。方法以CHO细胞DNA为模板,采用PCR扩增CHO细胞的MAR序列,克隆入逆转录表达载体PLXSN-CAT中,经限制性内切酶酶切及DNA序列分析鉴定目的基因。结果PCR扩增的特异性片段长度为476 bp,以此构建的重组质粒PLXSN-CAT-MAR经BamHⅠ酶切后显示为5.9 kb和476 bp左右的两条片段,测序结果与Gen-bank中的CHO细胞MAR基因（Genbank序列号M62716）序列一致。结论成功构建了CHO细胞MAR片段PLXSN-CAT-MAR重组逆转录表达载体。     

编码日本血吸虫大陆株磷酸丙糖异构酶(TPI)抗原基因的克隆与测序

目的 :获得编码日本血吸虫大陆株磷酸丙糖异构酶 ( Sjc TPI)的 c DNA基因片段 ,对其克隆与测序。方法 :以日本血吸虫大陆株成虫总 RNA为模板 ,经过逆转录 -聚合酶链 (式 )反应( RT- PCR)获得编码 Sjc TPI抗原的 c DNA片段 ,并克隆于载体 M13mp18、M13mp19,双脱氧链末端终止法测 DNA序列。结果 :体外 PCR扩增获得了编码 Sjc TPI的 0 .75kb c DNA片段 ,6个重组克隆子经酶切鉴定均有 0 .75kb片段的插入。测得编码 Sjc TPI的 DNA序列。结论 :用自行设计的引物成功地扩增了编码日本血吸虫大陆株 TPI抗原的基因片段并进行了克隆、测序 ,为制备重组 TPI进行疫苗试验打下基础。     

妊娠时间与肺结核复发的相关性研究及诊治对策

目的 分析肺结核史患者妊娠时间和肺结核复发间相关性.方法 选取我院收治的有肺结核史的妊娠妇女576例作为研究对象,对其妊娠前肺结核治疗、治愈后妊娠时间、妊娠后复发肺结核等进行分析,总结有肺结核史育龄女性的妊娠时间和肺结核复发之间的关系.结果 肺结核治愈后不同时间段妊娠者的结核复发率比较,差异具有显著性（P＜0.05）,停药后间隔时间越久妊娠,肺结核复发的几率越小.结论 加强孕期痰菌检查,及早发现复发肺结核,提高母婴安全.     

Effect of phosphorylation of MAPK and Stat3 and expression of c-fas and c-jun proteins on hepatocarcinogenesis and their clinical significance

AIM To study the effect of phosphorylation ofMAPK and Stat3 and the expression of c-fos andc-jun proteins on hepatocellular carcinogenesisand their clinical significance.METHODS SP immunohistochemistry was usedto detect the expression of p42/44MAPK, p-Stat3,c-fos and c-jun proteins in 55 hepatocellularcarcinomas (HCC) and their surrounding livertissues.RESULTS The positive rates and expressionlevels of p42/44MAPK, p-Stat3, c-fos and c-junproteins in HCCs were significantly higher thanthose in pericarcinomatous liver tissues (PCLT).A positive correlation was observed between theexpression of p42/44MAPK and c-fos proteins, andbetween p-Stat3 and c-jun, but there was nosignificant correlation between p42/44MAPK and p-Stat3 in HCCs and their surrounding livertissues.CONCLUSION The abnormalities of Ras/Rat/MAPK and JAKs/ Stat3 cascade reaction maycontribute to malignant transformation ofhepatocytes. Hepatocytes which are positive forp42/ 44MAPK, c-fos or c-jun proteins may bepotential malignant pre-cancerous cells.Activation of MAPK and Stat3 proteins may be anearly event in hepatocellular carcinogenesis.     

我国骨关节结核诊治的回顾与展望

骨关节结核是危害人们健康的严重感染性疾病,近95％由他处结核病继发而来.罹患骨关节结核疾病后几乎均将致残,严重影响人们的健康、工作和生活.建国以来在党和国家的关心和支持下,骨关节结核的诊治水平取得了长足进步.时至今日,由于多种原因,学科发展和被重视程度受到一定的制约,同整个医疗行业的发展不相适应.回顾过去,展望未来,我们需要重新审视骨关节结核的诊治方法,努力推进骨关节结核诊疗技术的科学发展.     

Replication and Inhibitors of Enteroviruses and Parechoviruses

The Enterovirus (EV) and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV). They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.     

心电图、心电向量图与冠状动脉造影结果对比分析

目的:通过分析心电图(Electrocardiogram,ECG)和心电向量图(Vectorcardiogram,VCG)的改变与冠脉造影（CAG）结果进行对比,探讨ECG、VCG在冠状动脉病变中的诊断价值。方法: 选择2008年1月~2009年12月临床拟诊断为冠心病患者108例,行常规ECG、VCG检查,并于1周内进行CAG,对检查结果依据各自的诊断标准进行判定,以CAG为标准诊断法,利用四格表法,计算相关评价真实性的指标并进行比较。结果: ①VCG检测的灵敏度、特异度、准确度显著高于ECG(P<0.05,P<0．01)。②ECG、VCG阳性率与冠脉病变支数组间比较:在单支病变、双支病变中,VCG阳性率明显高于ECG(P<0.05),左主干或三支病变无统计学意义;组内比较:ECG组左主干或三支病变组较单支病变、双支病变阳性率高(P<0.05,P<0.01);VCG组左主干或三支病变组较单支病变阳性率高(P<0.05);与双支病变阳性率比较无统计学意义;③ECG、VCG阳性率与冠脉病变程度组间比较:冠脉病变狭窄50%～69%的VCG阳性率明显高于ECG (P<0.05),其他两组阳性率比较无统计学意义;组内比较:ECG组冠脉病变狭窄≥90%较50%～69%、70%～89%的阳性率高(P<0.05,P<0.01); VCG组狭窄≥90%较50%～69%阳性率高（P<0.01),其他无统计学意义。结论: VCG对冠心病检测价值显著高于ECG。     

Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence

Here we report the structural characterization of the product formed from the reaction between hydroethidine (HE) and superoxide (O(2)(.-)). By using mass spectral and NMR techniques, the chemical structure of this product was determined as 2-hydroxyethidium (2-OH-E(+)). By using an authentic standard, we developed an HPLC approach to detect and quantitate the reaction product of HE and O(2)(.-) formed in bovine aortic endothelial cells after treatment with menadione or antimycin A to induce intracellular reactive oxygen species. Concomitantly, we used a spin trap, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), to detect and identify the structure of reactive oxygen species formed. BMPO trapped the O(2)(.-) that formed extracellularly and was detected as the BMPO-OH adduct during use of the EPR technique. BMPO, being cell-permeable, inhibited the intracellular formation of 2-OH-E(+). However, the intracellular BMPO spin adduct was not detected. The definitive characterization of the reaction product of O(2)(.-) with HE described here forms the basis of an unambiguous assay for intracellular detection and quantitation of O(2)(.-). Analysis of the fluorescence characteristics of ethidium (E(+)) and 2-OH-E(+) strongly suggests that the currently available fluorescence methodology is not suitable for quantitating intracellular O(2)(.-). We conclude that the HPLC/fluorescence assay using HE as a probe is more suitable [corrected] for detecting intracellular O(2)(.-).     

大鼠骨髓间充质干细胞的分离培养和外源基因的导入

目的探讨绿色荧光蛋白基因转染骨髓间质干细胞的可行性。方法采用F icoll-PaqueTMP lus淋巴细胞分离液,根据细胞密度梯度原理,分离大鼠骨髓间充质干细胞(rM SC s)并进行体外原代培养和传代扩增,倒置相差显微镜观察细胞生长情况,免疫细胞化学法对其初步鉴定。流式细胞仪分析转染效率。结果原代和传代培养的细胞呈现梭形外观,具有较强的生长增殖能力;细胞均一表达CD44、CD54、CD106、CD29抗原。电穿孔法转染rM SC s转染率为32.8%±3%。结论采用比重为1.077 g/L的F icoll-PaqueTMP lus能分离获得大鼠骨髓间充质干细胞,经原代培养和传代培养能够迅速扩增。电穿孔法具有较高的介导外源基因表达于rM SC s的效率。     

血清C-反应蛋白和红细胞分布宽度在AECOPD与稳定期的变化及意义

目的观察慢性阻塞性肺疾病(COPD)患者急性加重期入院、出院时血清C-反应蛋白(CRP)和红细胞分布宽度(RDW)与正常人的差异。方法分别检测健康老年组(年龄≥65岁)、COPD急性加重期患者入院、出院时CRP和RDW水平,比较组间各指标的差异。结果急性加重期患者入院、出院时CRP和RDW水平均高于健康老年组,入院时CRP水平高于出院时(P0.01),入院、出院时RDW水平无统计学差异。结论 CRP和RDW可一定程度评估COPD病情及反映病情变化。     

Effect of phosphorylation of MAPK and Stat3 and expression of c-fos and c-jun proteins on hepatocarcinogenesis and their clinical significance

AIM To study the effect of phosphorylation ofMAPK and Stat3 and the expression of c-fos andc-jun proteins on hepatocellular carcinogenesisand their clinical significance.METHODS SP immunohistochemistry was usedto detect the expression of p42/44~(MAPK), p-Stat3,c-fos and c-jun proteins in 55 hepatocellularcarcinomas (HCC) and their surrounding livertissues.RESULTS The positive rates and expressionlevels of p42/44~(MAPK), p-Stat3, c-fos and c-junproteins in HCCs were significantly higher thanthose in pericarcinomatous liver tissues (PCLT).A positive correlation was observed between theexpression of p42/44~(MAPK) and c-fos proteins, andbetween p-Stat3 and c-jun, but there was nosignificant correlation between P42/44~(MAPK) and p-Stat3 in HCCs and their surrounding livertissues.CONCLUSION The abnormalities of Ras/Raf/MAPK and JAKs/ Stat3 cascade reaction maycontribute to malignant transformation ofhepatocytes. Hepatocytes which are positive forp42/ 44~(MAPK), c-fos or c-jun proteins may bepotential malignant pre-cancerous cells.Activation of MAPK and Stat3 proteins may be anearly event in hepatocellular carcinogenesis.     

医源性消化道出血常见病因及其防治

目的分析医源性消化道出血的常见病因,并探讨防治措施。方法就临床所见的69例医源性消化道出血进行分析。结果医源性消化道出血分布于临床各科室,病因各不相同,但以外科手术、外伤、脑血管意外、。肾功能衰竭、呼吸功能衰竭、激素治疗、阿司匹林治疗等为主,临床上多以黑便为主要症状;对于已发生的医源性消化道出血,及时予制酸剂和胃黏膜保护剂治疗,能及时止血,大出血时经积极止血及抗休克治疗,甚至内镜治疗效果良好。结论医源性消化道出血各个临床科室均常见,病因各异,经积极、合理的治疗多预后良好。