Batroxobin,the thrombin-like enzyme,is used for therapeutic defibrination. We have found that batroxobin has good therapeutic effect in ischemic reperfusion rats and clinical practices in vivo. But we have not studied the neuroprotective effect of batroxobin on anoxic hippocampal neurons in vitro. The purpose of this study was to obtain further information on the mechanism of the batroxobin-induced neuroprotection and examine the neuroprotective effect on neurons exposed to anoxia. The effect of batroxobin on anoxic damages in cultured hippocampal neurons of neonatal rats was investigated by using morphological changes and heat shock protein 70Kd (Hsp70) immunoreactive expression as indicators. The results indicate that batroxobin, besides its defibrination, may have a direct neuroprotective effect on anoxic damage of hippocampal neurons.

丹参酮ⅡA磺酸钠抑制人肾间质成纤维细胞体外增殖过程中PCNA和Cyclin E的表达

丹参酮ⅡA磺酸钠(DS-201)是丹参脂溶成分丹参酮ⅡA经磺化而成的水溶性钠盐,药理作用与丹参酮ⅡA相同,因其水溶性提高、疗效强于丹参酮ⅡA而在临床心血管疾病的治疗和预防中广泛应用.在肾脏间质纤维化的治疗方面,其具体作用机制尚不清楚.本实验拟采用DS-201体外干预人肾间质成纤维细胞(hRIF),观察药物对细胞PCNA和CyclinE表达的影响,揭示DS-201治疗肾间质纤维化的分子机制.

人脂肪组织源性间充质干细胞分离与体外培养

各种疾病所导致的器官衰竭严重地危害着人类的健康,降低了患者的生活质量.干细胞所具有的自我复制和多向分化能力使它成为挽救器官衰竭、进行组织修复的理想种子细胞.在特定的条件下它可以分化为中胚层起源的多种组织细胞,如成骨细胞、软骨细胞、肌腱、脂肪细胞、血管内皮细胞、肌细胞及神经星状细胞等.目前干细胞主要来源于骨骼肌卫星细胞、骨髓干细胞、胚胎干细胞等,近几年国外有研究显示脂肪组织中也含有大量具有多向分化潜能的干细胞(adipose tissue-derived mesenchymal stem cells, ADMSCs),而目前国内应用脂肪组织分离及培养干细胞的报道极少.本研究拟将对ADMSCs进行分离和体外培养,并对其生长方式进行观察.

二硝基氟苯修饰的肿瘤疫苗可增强外周血单个核细胞体外杀伤人乳腺癌细胞

抗肿瘤免疫治疗是一种重要的癌症辅助治疗手段,其特点是毒副反应较小,患者耐受性好,并能在相当程度上改善肿瘤的预后.本研究是应用一种小分子半抗原二硝基氟苯(1)NP)修饰细胞瘤苗(TCV),评价该瘤苗是否会增强人淋巴细胞对人乳腺痛(MCF7)和人肺癌(H23)细胞的杀伤效应.此外,我们还把DNP瘤苗修饰法与另一种临床常用的瘤苗修饰法,即应用新城疫病毒Ulster株(NDV Ulster.)的修饰法,进行了抗肿瘤免疫效应方面的比较.

TGFβ1在体外培养人淋巴管内皮细胞的表达

内皮细胞具有活跃的代谢功能,血管内皮细胞合成和分泌功能已多有研究和评述,相比较而言,同属于内皮的淋巴管内皮细胞生物学功能却知之甚少.转化生长因子β1(TGFβ1)是一种多功能细胞因子,除了刺激多种细胞因子、炎性介质等合成分泌外,还影响肿瘤的形成和发展.近来研究表明淋巴管内皮细胞与肿瘤细胞的转移有关[1].本实验即以人淋巴管内皮细胞为研究对象,观察TGFβ1在其中的表汰.

三七总皂甙抑制兔血管平滑肌细胞增殖及促进C-MYC基因表达

血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的异常增殖与凋亡在动脉粥样硬化(atherosclerosis,AS)病理生理过程中起核心作用[1～3].三七总皂甙(panax notoginsengsaponins,PNS)具有抗AS的作用.本研究观察PNS对VSMCs增殖周期、细胞凋亡过程及凋亡相关基因C-MYC表达的影响,探讨其作用机制.

脂多糖对人脐静脉血管内皮细胞的直接损伤作用

血管内皮细胞(endothelial cells,EC)炎症反应是感染性休克向不良方向演变的重要原因,有关激活剂(LPS、IL-1)导致EC活化的研究是败血症病理反应的中心课题.内毒素引起微循环EC损伤是内毒素性组织损伤的重要环节之一.本实验探讨了LPS对体外培养脐静脉EC的直接损伤作用.

中药外用治疗滴虫性阴道炎69例临床观察

滴虫性阴道炎是妇科常见病,属于中医带下病"阴痒"范畴,传统中医治疗该病重内服,轻外治,因此往往不易根治.由于局部症状明显,并且主要病变在阴道穹窿部,宜选择散剂及洗剂.1996～2003年作者选用桔矾、土茯苓薰洗、坐浴及散剂外用,治疗滴虫性阴道炎69例,收到满意效果,现报道如下.

卡氏肺孢子虫肺炎动物模型及虫体体外培养研究

肺孢子虫(Pneumocystis)是一种机会性致病病原体,免疫功能受损的宿主感染后,可引起肺孢子虫肺炎(Pneumocystis pneumonia,PCP)或称肺孢子虫病(Pneumocystosis).长期以来,人们一直认为卡氏肺孢子虫(Pneumocystis carinii,Pc)是一种原虫,是引起人体"卡氏肺孢子虫肺炎"(Pneumocystis carinii pneumonia,PCP)的病原体.然而,20世纪80年代末的分子生物学和生物化学的研究结果证实,它属于真菌[1、2].引起人体肺孢子虫肺炎的病原体为Pneumocystis jeroveci(国内有学者译为"耶氏肺孢子虫"),而卡氏肺孢子虫仅感染大鼠和其他一些动物.鉴于目前国内对上述归类和新名尚缺乏统一认识,本文仍将之称为"肺孢子虫",暂不称"真菌".近年来,由于免疫抑制剂、抗肿瘤药物的广泛应用,以及世界各地AIDS病例的增加,PCP病例也随之急剧增多,使得肺孢子虫成为目前研究的热点.建立PCP动物模型和体外培养肺孢子虫,是开展肺孢子虫分类、生活史、发病机制、药物筛选、疫苗研制,以及免疫学与分子生物学等研究的基础.本文就卡氏肺孢子虫肺炎动物模型及其体外培养的研究进展综述如下.

中草药体外抑菌作用的研究进展

中药主要由植物药(根、茎、叶、果)、动物药(内脏、皮、骨、器官等)和矿物药组成.因植物药占中药的大多数,所以中药也称中草药.中草药是我国传统医学的重要组成部分.中国是中草药的发源地,目前我国大约有12 000种药用植物.目前,各地常使用的中药已达5000种左右,把各种药材相配伍而形成的方剂,更是数不胜数[1].

疫苗中和抗体体外检测方法的研究进展

中和抗体是抗病毒免疫的主要因素,也是疫苗免疫治疗效果的重要检测指标之一.预防疫苗的免疫保护性主要体现于是否可诱导出高滴度的中和抗体,因此中和抗体活性也是疫苗评估和质控的重要依据.迄今为止,根据已知抗原特性、抗原与抗体反应特点以及结果判定方法的不同,已建立了多种病毒中和抗体活性的体外测定方法.本文就近年来关于中和抗体活性体外测定方法的新研究进展做一综述.

卟吩姆钠漂白特性用于大鼠体外MLL细胞光动力学疗法治疗的单线态氧剂量估算

背景与目的光动力学治疗作用是靠单线态氧的生成介导.作者将基于动力学的单线态氧剂量模型用于光敏剂卟吩姆钠(pofimer sodium,Photofrin).Photofrin的漂白机制不仅有单线态氧介导的漂白,还有不依赖氧的漂白机制,作者试图将后一特点纳入Photofrin的单线态氧剂量模型中.

Purpose To assess the maximum uptake of Iododeoxyuridine (IUdR) by proliferating smooth muscle cells in vitro to determine the optimal concentration to be administrated in an in vivo experiment. The long-term goal is to utilize radioactive IUdR to inhibit smooth muscle cell proliferation and restenosis of arteries after balloon angioplasty in vivo. Methods Porcine smooth muscle cells (SMCs) were cultured in 5% FBS medium and stimulated to proliferate by the addition of medium containing 10% FBS and insulin. IUdR was added at 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, respectively, in proliferating SMCs with control for 1, 3, 5, 7 day incubation. Fluorescence Activated Cell Scanning (FACS) was performed after the SMCs were harvested and double-stained with FITC-conjugated anti-IUdR antibody (B44) and propidium iodide (PI). The ratio of IUdR-labeled cells to total cell population for each IUdR concentration and duration was determined by FACS. All data were repeated three times at each time point. The doubling times, growth curve and cell density of the proliferating SMCs were investigated using Beckman Coulter Particle Counter and digital microscopy. Results The percentage of proliferating SMCs uptaking IUdR increased from 1 to 5 days incubation with all concentrations of IUdR; In day 5, the uptake rate reached the peak value, then decreased by 7 days. IUdR uptake on day 5 was higher with concentrations of 10 μM and 20 μM. The doubling times of the SMCs were prolonged with IUdR concentration increasing, while the proliferating cell number and density compared with control decreased obviously by day 5 (P<0.05).Conclusion The peak time to uptake IUdR was 5 days and optimal concentration of IUdR was between10 μM to 20 μM for proliferating SMCs to uptake in vitro. IUdR itself could inhibit the SMCs' proliferation and the inhibitory effect was related to the concentration.

乳化异氟烷的体内和体外溶血性实验研究

Objective Emulsified isoflurane (8% ,vol/vol) is a kind of lipid based formation for intravenous administration. The purpose of this study is to evaluate whether emulsified isoflurane induces hemolysis or not in vitro and in vivo. Methods In hemolysis test in vitro, a male rabbit was used to prepare 2% (vol/vol) erythrocyte suspensions for measuring degree of hemolysis of emulsified isoflurane at the doses from 12 to 0. 3 g/L. In hemolysis test in vivo,4 male Beagle dogs were intravenously adminstrated emulsified isoflurane 225.6 mg/kg in 3-5 min. 5 ml samples of venous blood were collected from each dog at 0 min (start of injection) ,5 min,30 min,1 h,2 h,4 h,8 h,1 d,2 d,3 d and 6 d after the adminstration of emulsified isoflurance for measuring erythrocyte morphology,reticulocyte counts, the concentrations of free hemoglobin,haptoglobin,and bilirubin. At the same time, urinary blood, urinary bilirubin and urobilinogen were also measured before and after adminstration. Results In vitro experiment,emulsified isoflurane led to hemolysis at the concentrations from 12 to 1.2 g/L. However,no hemolysis was found at the concentrations from 0. 6 to 0. 3 g/L. In vivo experiment,with the exception of a slight reduction in indirect bilirubin and a mild increase in direct bilirubin at 5 min (P < 0.05 ) , others such as total bilirubin, retculocyte counts, haptoglobin, free hemoglobin,urinary bilirubin,urinary blood, and urinary urobinogen were not significantly different compared with their corresponding values before injection. There was also no significant difference in erythrocyte fragmentations at 0 min,5 min,30 min,1 h,2 h,4 h,8 h,1 d,2 d,3 d and 6 d after injection of emusified isoflurane, and none of macrocytes and nucleated red cells was noted on all blood films. Conclusions Emulsified isoflurane at concentrations recommended for clinical trials did not cause hemolysis in vitro and in vivo.

HCV IRES特异性抑制性RNA结构模拟及在体外对病毒翻译启动的抑制作用

[摘要]目的 计算机模拟IRNA二级结构,研究HCV IRES特异性抑制性RNA对HCVIRES介导蛋白翻译的体外抑制作用.方法 应用计算机软件模拟IRNA二级结构;体外化学合成IRNA及其互补体的cDNA,以AatⅡ和Xba Ⅰ双酶切后克隆入具有自剪切作用的顺式核酸载体pGEMRz中;应用酶切方法、PCR方法和测序法对重组载体进行三重鉴定.

多粘菌素B及其模拟肽体外抗内毒素的实验研究

目的:观察PMB及其模拟肽处理前后FITC-LPS与PMBC的结合能力及培养上清中IL-6、TNF α的含量变化.方法:LPS(或FITC-LPS)100μg/L分别与PMB100 mg/L、peptide 0 100 mg/L、peptide 1 100 mg/L、PBS,37℃孵育30 min后(分别加入50 mL/L NHS)刺激PBMC,用流式细胞仪检测FITC-LPS与PBMC的结合能力及CD14表达;ELISA法检测培养上清中细胞因子TNF α、IL-6含量.结果:FITC-LPS分别与PMB、peptidel孵育后,细胞膜平均通道荧光显著减少,LPS与PBMC的结合能力显著降低(分别为8.1±1.8 vs 15.8±4.5 P＜0.01;8.5±2.0vs15.8±4.5 P＜0.01).100μg/L LPS刺激PBMC3h后CD14阳性率明显增加;LPS分别与PMB和peptidel预孵育后可显著降低LPS刺激PBMC细胞膜CD14表达(分别为45.5±6.2%vs68±5.5%P＜0.01;43.2±4.1%vs68±5.5%P＜0.01).LPS刺激PBMC分泌TNF-α和IL-6显著增加,LPS分别与PMB和peptidel预孵育后能显著减少细胞因子TNF-α(分别为15.30±1.0vs45.9±5.7 P＜0.01;18.2±0.9vs45.9±5.7P＜0.01)和IL-6分泌(分别为50.5±4.2 vs176.4±12.1 P＜0.01;58.1±4.1 vs176.4±12.1 P＜0.01).结论:多粘菌素B及其模拟肽(Peptidel)可能通过下调PBMC CD14表达,降低细胞因子水平来减少LPS诱导的炎症反应.

HGF和FGF4体外诱导人骨髓CD45-CD117-细胞向肝细胞分化的研究

目的:探讨在HGF、FGF4诱导下人骨髓CD45-CD117-细胞能否向肝细胞分化,并探讨两种生长因子在诱导分化中可能的作用机制.方法:骨髓来自4名4-40岁健康志愿者的胸骨或髂骨.用两步磁式分离法分离出CD45-CD 117-细胞,分别用含有FGF4,HGF,HGF+FGF4或不加生长因子的DMEM培养基进行培养,相应地分为A,B,C,D四组.分别于新分离时和培养7 d,14 d,21 d,28 d时留细胞.备作免疫细胞化学检测AFP,CK18,白蛋白,PAS染色检测糖原等肝系细胞的特征型标志,RT-PCR检测C、D组细胞c-met(HGF受体)和FGFR2(FGF4R)mRNA表达情况.结果:A,B,C三组加生长因子诱导培养后的细胞均可检测到肝系细胞的标志,D组细胞不加生长因子培养后没有检测到肝系细胞的标志.c-met和FGFR2mRNA在新分离的和D组培养7 d,14 d时的细胞均有较弱的表达,而在C组细胞诱导培养7 d,14 d时表达升高.结论:HGF和FGF4可诱导人骨髓CD45-CD117-细胞向肝细胞样细胞分化.生长因子诱导分化的作用可能通过与其受体之间的正反馈调节.

细菌内同源重组法构建HBV S区和C区基因非复制型腺病毒载体及其体外表达

目的:构建表达乙肝病毒表面抗原(HB sAg)和e抗原(HBeAg)的非复制型重组腺病毒载体,并检测他能否在真核细胞中有效表达目的基因.方法:扩增乙肝病毒(HBV)前S2/S基因和前C/C基因片段,分别亚克隆到腺病毒穿梭质粒pAdTrack-CMV上,与5型腺病毒骨架质粒pAdeasy-1共转染BJ5183细菌,经细菌内同源重组产生分别携带HBV S区和C区基因的重组腺病毒载体pAd-HBs和pAd-HBe,经脂质体法转化293细胞包装产生重组腺病毒Ad-HBs和Ad-HBe;体外转染293和Vero细胞,RT-PCR和ELISA法检测目的基因的表达.结果:构建了表达HBsAg和HBeAg基因的重组腺病毒,病毒滴度可达5x1012pfu/L,并能在真核细胞中有效表达目的基因.结论:成功构建表达HBsAg和HBeAg的重组腺病毒载体,为进一步开展HBV基因治疗研究提供实验基础.

诱导HSP70减轻肠上皮细胞缺氧/再给氧的损伤

目的:观察热休克预处理诱导热休克蛋白70(HSP70)对肠上皮细胞(IEC-6)缺氧/再给氧损伤的影响,并探讨HSP70的细胞保护作用.方法:体外培养IEC-6细胞,随机分为正常对照(C)、单纯缺氧/再给氧(H/R)及热休克预处理组(HS+H/R),免疫组化检测缺氧/再给氧后各组细胞HSP70表达程度,MTT法分析细胞活力,并检测细胞LDH漏出,TUNEL法检测细胞凋亡情况.结果:与C组及H/R组比较,热休克预处理可明显诱导HSP70的产生(0.93±0.07 vs 0.20±0.04,0.39±0.08,P＜0.01),热休克预处理可显著增加IEC-6细胞缺氧/再给氧处理后细胞活力(0.27±0.04 vs 0.24±0.02,P＜0.05),LDH漏出减少(1021.29±207.06 vs 1419.90±393.07,P＜0.05),细胞凋亡率也明显降低(7.8±1.6% vs 12.3±2.9%,P＜0.05).结论:通过热休克预处理诱导HSP70表达,可明显减轻肠上皮细胞缺氧/再给氧损伤.防止肠上皮细胞缺氧/再给氧后细胞凋亡可能是其保护作用机制之一.