反转录巢式PCR方法检测SARS冠状病毒

目的探索SARS冠状病毒早期诊断的有效手段。方法对临床病例样品采用反转录巢式PCR方法鉴定SARS冠状病毒。提取样品RNA,用巢式PCR外侧下游引物进行反转录,以反转录产物为模板,进行巢式第一次、第二次PCR。PCR产物克隆到pMD18-T载体后进行测序鉴定。结果被检样品扩增出特异片段,经测序验证确实来自SARS相关冠状病毒。Blast比较结果与SARS冠状病毒多伦多株相应片段只有一个碱基的差异。结论反转录巢式PCR方法验证在非典型肺炎患者标本中存在SARS相关冠状病毒的序列,证实该法不失为一种检测SARS冠状病毒的快速、简便、易行的方法。     

高压氧对脂多糖内毒素致急性肺损伤大鼠的抗炎作用

目的 探讨高压氧(HBO)对脂多糖内毒素致急性肺损伤大鼠的抗炎作用及治疗效果.方法 选择46只Sprague-Dawley大鼠,雌雄各半,分为空白组10只、模型组12只、HBO治疗组24只.模型组和HBO治疗组采用尾静脉注射内毒素(5 mg/kg体重)制作大鼠急性肺损伤模型;HBO治疗组大鼠造模后给予HBO治疗,压力0.2 Mpa(2.0 ATA),进舱后洗舱5 min,升压5 min,稳压吸氧40 min,减压10 min出舱;空白组注射同等剂量生理盐水.各组分别于处理后第3小时和第6小时进行白细胞计数、动脉血气分析、白细胞介素8(IL-8)水平检测及计算肺湿重/干重比等,并在光镜下观察肺组织病理变化.结果 HBO治疗组与模型组比较,血氧分压提高,肺湿重/干重比、白细胞及中性粒细胞数、IL-8水平均下降,病理组织切片可见炎性细胞浸润较少.结论 HBO治疗急性肺损伤,不仅可以改善组织缺氧,还可产生一定的抗炎作用,从而对急性肺损伤发挥疗效.

Abstract：

Objective To investigate any anti-inflammatory and curative effect of hyperbaric oxygen (HBO) therapy on mice with acute lung injury (ALI) induced by lipopolysaccaride endotoxin. Methods Fortysix Sprague-Dawley rats, half male and half female, were randomly divided into a normal saline group with 10 rats,an LPS group with 12 rats, and an HBO group with 24 rats. LPS was injected via the tail vein at 5 mg/kg weight to establish the ALI model. The HBO group rats were treated with HBO. Then leucocyte counts, arterial blood gas analysis, interleukin-8 (IL-8) in the blood and lung wet/dry weight (W/D) were compared among the 3 groups at the 3rd and 6th hours. Pathological changes were examined under a light microscope. Results Compared with the LPS group, blood oxygen and W/D were higher in the HBO group, the white blood cell, neutrophil and IL-8 all were lower, and inflammatory cell infiltration was less in lung slides from the HBO group. Conclusions HBO therapy can improve oxygen deficiency and has an anti-inflammatory effect. As a result it might have protective effect against ALI.     

痘苗病毒寡核苷酸检测芯片的设计及研制

目的对痘苗病毒进行寡核苷酸检测芯片的初步研究,为建立寡核苷酸芯片检测该类病毒提供初步研究依据。方法根据痘苗病毒特异基因设计寡核苷酸探针,人工合成探针后制备寡核苷酸芯片。在病毒感染的不同阶段提取病毒样品DNA及阴性样品DNA,采用限制性显示技术标记,标记样品与芯片杂交后,用Agilent芯片扫描仪检测杂交结果。结果芯片与病毒样品杂交有较强的杂交信号,而与阴性样品杂交除阳性探针外均无信号。结论病毒样品与阴性样品杂交信号区别明显,在病毒感染的各个时段也都有明显的杂交信号,反映了寡核苷酸芯片具有较高的特异性和灵敏度。     

阿司匹林预处理对原代培养大鼠Ⅱ型肺泡上皮细胞抗氧化损伤的影响研究

目的 观察阿司匹林对原代培养大鼠Ⅱ型肺泡上皮细胞(AEC Ⅱ)的保护效应,并探讨其抗氧化损伤的机制.方法 将原代分离、纯化、培养的离体大鼠AEC Ⅱ分为5组.过氧化氢损伤(H2O2)组在培养40 h后加入0.5 mmol/L H2O2,建立细胞氧化损伤模型;生理盐水(NS)组则加入NS ;阿司匹林预处理1、2、3(A1～3)组在加入H2O2前给予阿司匹林50、100、200μmol/L预处理.3 h后观察细胞形态学变化和贴壁细胞计数;采用四甲基偶氮唑盐(MTT)比色法检测细胞存活率;采用免疫组化法和聚合酶链反应法检测NS组、A1～3组在培养20、40、60 h AEC Ⅱ中血红素氧合酶-1(HO-1)的蛋白及mRNA表达.结果 采用胰蛋白酶消化、免疫黏附法每只鼠可收获(2.0～2.5)×107个AEC Ⅱ,纯度和活性均＞90%.与NS组比较,H2O2组细胞间隙增宽,贴壁细胞数减少,细胞皱缩,细胞存活率(A值)明显下降(0.054 6±0.004 0比0.103 8±0.009 9,P＜0.01);与H2O2组比较,A1～3组贴壁细胞数增多,细胞形态较完整,无明显皱缩,细胞存活率(A值)明显增加(0.066 9±0.003 9、0.071 0±0.006 5、0.078 7±0.009 2比0.054 6±0.004 0,均P＜0.01).与NS组比较,A1～3组培养20、40、60 h时HO-1的蛋白及mRNA表达均明显增加,60 h时达峰值[蛋白(积分A值):1.59±0.12、1.60±0.09、1.61±0.08比1.25±0.11;mRNA(Ct值比值):24.31±1.74、30.45±2.53、32.63±3.74比22.99±1.95,均P＜0.05];但A1～3组间HO-1蛋白表达无明显差异.结论 阿司匹林通过上调HO-1表达对离体培养氧化损伤的大鼠AEC Ⅱ起保护效应;HO-1可能是其中重要的保护调节因子.

Abstract：

Objective To investigate the protective effect of aspirin on primary cultured type Ⅱ alveolar epithelial cell (AEC Ⅱ ), and the mechanism of its effect on anti-oxidation damage. Methods The original generation of adult rat AEC Ⅲ were cultured and purified. They were divided into normal saline (NS) group,hydrogen peroxide injury group (H2O2 group), and 1, 2, 3 aspirin pretreatment groups (Al -3 groups). In H2O2 group, 0. 5 mmol/L H2O2 was added to AEC Ⅱ after 40 hours of culture to reproduce a cell oxidative injury model. In NS group, only NS was added to AEC Ⅱ culture. To the A1 - 3 groups aspirin 50, 100 and 200 μmol/L were added respectively. Cell form, cell count and cell survival rate were observed at 3 hours after H2O2 was given. Immunohistochemical and polymerase chain reaction (PCR) methods were used for the determination of heme oxygenase-1 (HO-1) protein and HO-1 mRNA (20, 40, 60 hours of culture). Results With trypsin digestion and immune adherence method AEC Ⅱ could be harvested (2.0- 2. 5)× 107, and the purity and activity were both over 90%. Compared with NS group, gaps between cells were widened in H2O2 group, cell account was reduced, and the survival rate (A value) was reduced significantly (0. 054 6±0. 004 0 vs. 0. 103 8±0. 009 9, P＜0. 01). Compared with H2O2 group, in A1 - 3 groups the number of adherent cells was increased, cell morphology was intact, and no obvious cell shrinkage was found. Higher survival rate (A value) was found in A1 - 3 groups than that of H2O2 group (0. 066 9±0. 003 9, 0. 071 0±0. 006 5,0. 078 7±0. 009 2 vs. 0. 054 6±0. 004 0, all P＜0. 01). Compared with NS group, HO-1 protein and HO-1 mRNA expression in AEC Ⅱ after 20, 40 and 60 hours of culture reached peak level at 60 hours, and they were increased significantly in A1 - 3 groups [protein (A value) : 1.59±0. 12, 1.60±0. 09, 1.61±0. 08 vs.1.25±0. 11; mRNA (the ratio of Ct value: 24.31±1.74, 30. 45±2. 53, 32. 63±3. 74 vs. 22.99±1.95, all P＜0. 05]. There was no significant difference in HO-1 protein expression among A1 - 3 groups. Conclusion There are significant protective effects of aspirin against anti-oxidative damage in cultured AEC Ⅱ cell.As expression of HO-1 is increased in aspirin groups, it may be considered as a protective factor agninst anti-oxidative damage in AEC Ⅱ cell culture.     

乙型肝炎病毒转基因小鼠树突状细胞提呈功能的初步研究

目的;探讨HBV转基因小鼠树突状细胞（DC）功能与其免疫耐受状态的相羞生。方法：按改良Steinman方法,对正常非转基因小鼠和HBV转基因小鼠脾脏树突状细胞进行分离,DC经HBeAg预刺激后,再用此DC与ConA共刺激淋巴细胞增殖反应。结果：HBV转基因小鼠的DC细胞数为10^2／孔、10^3／孔、10^4／孔,分别刺激各组小鼠淋巴细胞时,HBV转基因小鼠及非转基因小鼠的淋巴细胞^3H-TdR掺入量均显著低于对照组。结论：提示HBV转基因小鼠的DC提呈功能低下。     

体外冲击波碎石治疗肾结石的临床疗效观察

目的:评估体外冲击波碎石治疗肾结石的临床效果。方法:对2006年8月～2008年8月间体外冲击波碎石治疗的126例肾结石患者作回顾性分析。术后3个月评估效果,结石完全排出或残余结石0.4cm为治疗成功,未排或残余结石0.4cm则为治疗失败。结果:经体外冲击波碎石治疗的126例肾结石病人中,109例在3个月内排尽结石,13例残留结石,4例碎石无效。结论:体外冲击波碎石是治疗肾结石的首选方法,安全有效,是治疗肾结石值得选择的方法。     

雾化吸入氟化碳对脂多糖性肺损伤大鼠肺内AQP-1及钠钾ATP酶活性的影响

目的观察雾化吸入氟化碳（perfluorocarbon,PFC）对急性肺损伤（acute lung injury,ALI）大鼠肺水肿、肺内水通道蛋白-1（AQP-1）及钠钾ATP酶活性的影响。方法大鼠腹腔注射脂多糖（lipopolysaccharide,LPS）建立ALI模型并雾化吸入PFC进行干预。在LPS注射后6h观察PFC对ALI大鼠动脉血气、肺损伤评分、肺湿干质量比值、肺泡通透指数及钠钾ATP酶活性等的影响,并采用western-blot观察肺内AQP-1表达的变化。结果LPS性肺损伤以肺水肿为主要表现之一。雾化吸入PFC可以缓解LPS诱导ALI后的低氧血症,降低ALI大鼠的肺损伤评分、肺湿干质量比值、肺泡通透指数,同时增加肺组织钠钾ATP酶活性及AQP-1的表达。结论雾化吸入PFC减轻LPS性肺损伤大鼠肺水肿,上调肺损伤期间钠钾ATP酶活性与AQP-1表达。     

广东省食品中单核细胞增多性李斯特菌脉冲场凝胶电泳技术分子分型的研究

目的应用脉冲场凝胶电泳技术（PFGE）对广东省食品中分离的单核细胞增多性李斯特菌（Lm）进行分子分型,并建立PFGE数据库。方法参照美国PulseNet的Lm PFGE分子分型标准方法进行检测。应用BioNumerics软件对不同食物种类、时间和地点的分离株进行比对,分析菌株之间的相关性。结果107株Lm分成41个PFGE型,型别较为分散;从鸡肉中分离的菌株数最多（37株）,PFGE分型也最多（19个）;冷藏和冷冻食品的分离率较高,其中有26株菌仅存在1-2个电泳条带的差异,相似度极高;韶关和惠州地区分离的Lm存在着地区特异性;随时间变迁,部分菌株仍存在不同程度的相关性。结论广东省食品中分离的Lm在整体上表现出较大的遗传多样性,但部分菌株有不同程度的相关性。