双歧杆菌减轻肠外营养幼兔肝损害的实验研究

目的长期肠外营养(PN)患儿容易发生肝损害并发症,如肝脂肪变、胆汁淤积,甚至肝衰竭,其机制仍不清楚,可能和肠屏障功能障碍有关。近来双歧杆菌作为一种益生菌,在调节肠道微环境、保护肝脏中的作用日益受到重视。本研究拟通过给PN幼兔添加双歧杆菌,探讨其保护机制。方法生后3周的新西兰种白兔27只,体重200~250g。分为3组,PN组10只,PN+双歧杆菌组8只,对照组9只。双歧杆菌组每日经胃管注入丽珠肠乐溶液1ml/只(含青春双歧杆菌0.5×108),PN组注入生理盐水1ml/只。PN持续10d,取血测肝功能、内毒素水平;作肝脏组织病理分级评分;作回肠黏膜显微测量;作内脏组织匀浆和血培养观察细菌移位。结果双歧杆菌组幼兔肝功能明显改善,血清总胆红素和胆汁酸含量较PN组显著下降(P<0.05)。病理切片显示双歧杆菌组幼兔肝小叶完整,细胞形态基本正常,个别存在轻度炎症细胞浸润和纤维组织增生。而PN组则出现明显肝细胞变性(主要为脂肪变性)、胆管增生和胆汁淤积。参照Loff肝脏病理学评分标准,显示PN组分值明显高于对照组和双歧杆菌组(P<0.05),而双歧杆菌组和对照组比较差异无显著性意义(P>0.05)。肠道病理切片的计算机显微测量结果显示,双歧杆菌组幼兔回肠绒毛高度、隐窝深度、绒毛面积显著高于PN组(P<0.05),和对照组比较差异无显著性意义(P>0.05)。PN组幼兔血浆内毒素水平显著升高(P<0.001),双歧杆菌组内毒素水平处于正常范围。内脏组织、器官细菌培养结果显示,PN组幼兔肠菌移位率明显高于双歧杆菌组(P<0.01)。结论双歧杆菌可能通过降低肠黏膜通透性,避免幼兔产生肠菌移位和内毒素血症,保护肝功能。     

卡介苗多糖核酸与减毒活卡介苗治疗哮喘患儿疗效比较及治疗前后细胞因子的变化

目的探讨卡介苗多糖核酸（BCG-PSN）与减毒活卡介苗治疗儿童哮喘临床疗效及其对哮喘患儿TH1／TF12调节的差异。方法急性发作期哮喘患儿60例随机分A、B、C3组，每组20例。A组予全球哮喘防治创议（GINA方案）＋BCG-PSN0．5mg／次，肌注，1次偶日，共36次；B组予GINA方案＋减毒活卡介苗0．1mL，皮内注射，仅1次；C组单纯GINA方案治疗。酶联法检测治疗前后血清IgE及外周血单个核细胞（PBMC）诱生的IL-4、IL-5及IFN-γ。结果1．疗效：A组优于B组，B组优于C组（P均〈0．05）。2．治疗组治疗后IFN-γ均高于治疗前，IL-4、IL-5、IgE均低于治疗前。对于调整IL-5，A组优于B组和C组（P〈0．05，0．01）；对于上调IFN-γ及下调IL-4、IgE，A与B组比较无显著差异（P〉0．05）。结论1．BCG—PSN的疗效优于减毒活卡介苗。2．减毒活卡介苗一次性小剂量接种对哮喘儿IL-4、IFN-γ、IgE失衡的调节与BCG-PSN多次肌注效果相当，对IL-5失衡的调节不及BCG—PSN。     

血液透析与血液透析滤过急性肾衰竭患儿治疗前后血清P2微球蛋白下降率对比

目的探讨血液透析（HD）及血液透析滤过（HDF）治疗急性肾衰竭患儿前后血清陉微球蛋白（β2-MG）及下降率的影响。方法对各种原因导致的急性肾衰竭需行血液净化治疗38例患儿随机分成2组。HD组20例，HDF组18例。透析前后分别测血清β2-MG水平。结果HDF与HD前后血清陉．MG变化经统计学处理。HDF组治疗前后血清β2-MG水平差异有显著性意SL（P〈0．01）；HD组治疗前后血清β2-MG水平差异无显著性意义（P〉0．05）；HDF和HD组治疗后比较血清β2-MG水平差异、下降率差异均有显著性意义（P均〈0．01）。结论HDF清除血β2-MG明显优于HD。     

来氟米特对大鼠被动型Heymann肾炎的治疗作用

目的：研究来氟米特对被动性Heymann肾炎（PHN）的治疗作用,为临床膜性肾病的治疗提供依据.方法：Wistar大鼠尾静脉注射抗FX1A血清,建立PHN模型;用BCA法测量24h尿蛋白变化,用光镜、电镜观察肾脏病理改变,并用RT-PCR观察nephrin mRNA表达改变.结果：成功复制了PHN模型来氟米特能减轻PHN大鼠蛋白尿,肾脏病变程度,抑制nephrin mRNA表达减少.结论：来氟米特对PHN有一定的治疗作用,可能是通过提高nephrin表达及减少免疫复合物沉积来实现这一作用.     

Construction of eukaryotic expressing plasmids encodingHA andHA 1 of influenza A virus and their transient expression in HEK293 cells

Summary In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encodingHA andHA ₁ of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells.HA andHA ₁ genes were amplified by RT-PCR and cloned into pcDNA3. 1(+) to generate pcDNA3. 1(+)/HA and pcDNA3.1(+)/HA₁, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3. 1(+)/HA and pcDNA3.1(+)/HA₁ were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3.1(+)/HA or pcDNA3.1(+)/HA₁. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA₁, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine. Zhang Weidong, female, born in 1970, M. D., Ph. D. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 39970830).     

Expression and characterization of Gag protein of HIV-1（CN） in Pichia pastoris

To express the core protein of HIV-1 of Chinese prevalent strain （HIV-1 （CN）） in Pichia pastoris, the fulllength gag gene was inserted into the secretory expression vector pHILS1. Linearized recombinant plasmid pHILGAG by Sail was electrotransformed into the yeast strain GS115, and the yeast transformants were identified by PCR. To induce the interest protein to be expressed, the PCR positive transformants were inoculated in the medium of BMGY and BMMY, mRNA of the strain was detected by RT-PCR, and the expressed protein was analyzed by SDS-PAGE, Western blotting and thin layer scanning. mRNA （1.3 kb） was amplified by RT-PCR. SDS-PAGE and Western blotting analysis showed that the molecular mass of the expressed protein was 55 kD, which was similar to the expected value, and the expressed protein could react with McAb to HIV-1 p24. Thin layer scanning analysis demonstrated that the whole amount of the expressed protein was approximately 13 % of the soluble protein in the supernatant. The recombinant yeast had good genetic stability. The optimal expression conditions of the engineering yeast were as follows： BMMY medium, 80-90% of dissolved oxygen, 1% methanol, and 3-day-cultivation course. Gag proteins were expressed under the optimal expression condition and purified via gel filtration chromatography. The purity of the interest protein was up to 85 %. After the purified proteins were inoculated into BALB/c mice, the anti-HIV-1 antibodies in the immunized mice could be detected by Western blotting.     

DNA vaccination using bacillus Calmette-Guerin-DNA as an adjuvant to enhance immune response to three kinds of swine diseases

In order to enhance the immune efficacy of DNA vaccination, experiments were conducted to investigate the regulating effects of Bacillus Calmette-Guerin （BCG）-DNA as an adjuvant on immune responses of mice against foot-and-mouth disease （FMD）, Aujeszky＇s disease （AID） and classical swine fever （CSF）. BCG-DNA was purified from BCG by ion-exchange chromatography. Three DNA vaccines （pVSG, pVgD and pVE2） against the respective infection were constructed, and BCGDNA was coimmunized to mice by muscle injection. The results showed that titres of specific immunoglobulin （Ig）G to the vaccines mounted remarkably in the sera of the adjuvant covaccinated mice （P〈0.01）. Antibody isotype IgG2a and IgG1 also increased, respectively, in mice coimmunized with BCG-DNA compared with those of the control groups （P〈0.01）. Cellular immune cytokine interferon-gamma and cytotoxic T lymphocytes were detected in coimmunized BCG-DNA groups （P〈0. 05）. Whereas interleukin-4, humoral immune cytokine, was not significant （P〉 0. 05）. These results suggest that codelivery of BCG-DNA with DNA vaccines against FMD, AjD and CSF can enhance the induction of antigen-specific, especially, cell-mediated immunity.     

In vitro and in vivo antifungal activities of the eight steroid saponins from Tribulus terrestris L. with potent activity against fluconazole-resistant fungal pathogens

Antifungal activity of natural products is being studied widely. Saponins are known to be antifungal and antibacterial. We have isolated eight steroid saponins from Tribulus terrestris L. , namely TTS-8, TTS-9, TTS-10, TTS-11, TTS-12, TTS-13, TTS-14 and TTS-15. TTS-12 and TTS-15 were identified as tigogenin-3-O-β-D-xylopyranosyl（1→ 2）-[-β-D-xylopyranosyl（ 1 → 3 ） 3-β- D-glucopyranosyl （ 1 → 4 ）- 1- α-L-rhamnopyranosyl （ 1 → 2 ） 3-β-D-galactopyranoside and tigogenin-3-O-β-D-glucopyranpyranosyl（1→2）-[-β-D-xylopyranosyl（1→ 3）3-β-D-glucopyranosyl（1→4）-β-D-galactopyranoside, respectively. The in vitro antifungal activities of the eight saponins against six fluconazole-resistant yeasts, Candida albicans, Candida glabrata, Candida para psilosis , Candida tropicalis , Candida krusei , and Cryptococcus neo f ormans were studied using microbroth dilution assay. The results showed that TTS-12 and TTS-15 were very effective against several pathogenic candidal species and C. neoformans in vitro. It is noteworthy that TTS-12 and TTS-15 were very active against fluconazole-resistant C. albicans （MIC80 = 4.4, 9.4 mg/ml）, C. neoformans （MIC80 =10.7, 18.7 mg/ml） and inherently resistant C. krusei （MIC80 =8.8, 18.4 mg/ml）. So in vivo activity of TTS-12 in a vaginal infection model with fluconazole-resistant C. albicans was studied in particular. Our studies revealed TTS-12 also showed in vivo activities against fluconazole-resistant yeasts. In conclusion, steroid saponins TTS-12 and TTS-15 from Tribulus terrestris L. have significant in vitro antifungal activity against fluconazole-resistant fungi, especially TTS-12 also showed in vivo activity against fluconazole-resistant C. albicans.     

上海市虹口区预防接种不良反应主动与被动监测情况分析

目的：为了解预防接种不良反应主动与被动监测情况，为AEFI监测体系提供科学依据。方法：采用对照比较的方法分析虹口区2005年1-9月主动与被动监测预防接种不良反应情况。结果：主动监测的发热、硬结、红肿反应发生率为731．53／10万、414．53／10万、170．69／10万显著高于被动监测73．78／10万、8．20／10万、16．40／10万，主动监测的过敏性皮疹反应率105．67／10万，显著高于被动监测6．56／10万。门诊自身前后比较，4-9月主动监测的发热、硬结、红肿反应发生率显著高于1-3月28．06／10万、56．12／10万、28．06／10万；1-3月被动监测异常反应为0,4-9月主动监测异常反应发生率为113．79／10万。结论：主动监测预防接种不良反应，敏感性高，特异性好，对于完善国家对异常反应的经济补偿政策：正确评估接种疫苗后可能发生的不良反应事件．并提出预防和处理建议．都具有重要意义。     

槐果碱体外抗柯萨奇病毒B3m的作用

目的 以HeLa细胞为模型,观察从苦参中提取的槐果碱在体外抗柯萨奇病毒B3m(CVB3m)的作用.方法 ①用微量细胞培养法观察槐果碱对HeLa细胞的毒性.②用细胞病变效应(CPE)抑制法观察槐果碱体外抗CVB3m作用.③用MTT法和结晶紫染色法观察槐果碱对CVB3m感染的HeLa细胞的保护作用:在96孔板上种植HeLa细胞并予CVB3m吸附1 h,加入不同浓度的槐果碱,并设立病毒、细胞、槐果碱对照,培养15 h后以MTT法和结晶紫染色法测定比较各组细胞能量代谢率和细胞存活数.结果 ①槐果碱稀释到＜391 μg/mL,对HeLa细胞无毒性;≥783 μg/mL引起HeLa细胞CPE.②槐果碱6.25 ～ 50 μg/mL有直接抗病毒作用,减轻CPE;＞100 μg/mL反而会加重、加快CPE.③槐果碱1.56 ～ 25 μg/mL对感染CVB3m的HeLa细胞有保护作用,用MTT法和结晶紫染色法测得细胞能量合成代谢、细胞存活数较病毒对照组增加(P＜0.05);槐果碱在50、100 μg/mL时反而加重病毒对细胞的抑制,使细胞存活数、细胞代谢率较病毒对照组下降(P＜0.05).结论 一定浓度的槐果碱在体外有抗CVB3m作用,对感染CVB3m的HeLa细胞有保护作用.