局灶脑缺血再灌注期亚低温对神经元凋亡及Bcl－2和Bax表达的影响

目的：研究再灌注期实施亚低温治疗对大鼠大脑中动脉闭塞(MCAO)后神经元细胞的凋亡及Bcl-2，Bax基因表达的影响。方法：采用Longa线栓法制作MCAO模型，模型成功后分别进行缺血期3h、再灌注期3h、缺血期至再灌注期6h的亚低温治疗。再灌注后24h断头取脑，连续切片作TUNEL染色及Bcl-2，Bax免疫组化染色。结果：①与对照组的Bcl-2阳性细胞百分率[(19．79&;#177;0。92)％]相比，缺血期亚低温3h组[(23．04&;#177;3．76)％]和缺血亚低温至再灌亚低温6h组[(25．47&;#177;2．03)％]的Bcl-2阳性细胞率增加(q=4．19,0．O1&;lt;P&;lt;O．05；q=7．17，P&;lt;0．01)。比较单纯再灌亚低温3h组[(22．21&;#177;2．25)％]与缺血亚低温至再灌亚低温6h组的Bcl-2阳性细胞百分率，前者Bcl-2阳性细胞率较后者低(q=4．1l，P&;lt;0．01)。②与对照组的Bax阳性细胞百分率[(52．15&;#177;6．18)％]相比，缺血期亚低温3h组[(32．35&;#177;3．71)％]和缺血亚低温至再灌亚低温6h组[(26．86&;#177;2．43)％]的Bax阳性细胞百分率均降低(q=14．21，P&;lt;0．O1；q=18．95，P&;lt;0．01)；而单纯再灌亚低温3h组的Bax阳性细胞百分率[(47．26&;#177;4．52)％]降低不明显(q=2．75，P&;gt;0．05)。③凋亡细胞阳性率与Bcl-2／Bax的比值呈负相关关系(r=-O．9759，P&;lt;O．01)。结论：①单纯于再灌注期实施亚低温3h不能明显抑制神经元细胞凋亡，对Bcl-2和Bax基因的表达也无明显影响；但将缺血期实施的亚低温延长至再灌注期后3h能进一步抑制神经元细胞凋亡并降低Bax基因的表达。②Bcl-2／Bax的比值能影响细胞凋亡的发生。     

亚低温对大鼠局灶性脑缺血神经元细胞凋亡及调控基因Bcl-2和Bax表达的影响

目的研究不同时程的亚低温治疗对大鼠大脑中动脉闭塞(MCAO)模型的神经元细胞凋亡及Bcl-2,Bax基因表达的影响。方法健康雄性SD大鼠共40只,月龄两三个月。按抽签法随机分为5组,每组8只(n=8),分别为:假手术组(A)、对照组(B)、亚低温0.5h组(C)、亚低温1.0h组(D)、亚低温3.0h组(E)。采用Longa线栓法制作MCAO模型,缺血3.0h后再灌注。缺血后即刻分别进行不同时程(0.5,1.0,3.0h)的亚低温治疗,再灌注后24h断头取脑,连续切片作TUNEL染色及Bcl-2,Bax免疫组化染色。结果凋亡神经元细胞(TUNEL阳性细胞)主要见于梗死灶周围的纹状体及额顶部皮层。Bcl-2及Bax阳性细胞也主要见于梗死灶周围的纹状体及额顶部皮层,与该区凋亡细胞的集中分布相一致。与对照组的Bcl-2细胞阳性率相比,亚低温3.0h组增加犤(23.0±3.8)%,P<0.05)犦;与对照组的Bax细胞阳性率相比,亚低温1.0,3.0h组均降低犤分别为(45.9±4.1)%,(32.4±3.7)%,P<0.05,P<0.01犦;凋亡细胞阳性率与Bcl-2/Bax的比值呈负相关关系(r=-0.9759,P<0.01)。结论亚低温1.0h以上有抑制细胞凋亡作用;Bcl-2/Bax的比值能影响细胞凋亡的发生。     

全脑缺血再灌注损伤大鼠海马神经元凋亡:调节蛋白Bcl-2和Bax的表达(英文)

背景:脑缺血再灌注对中枢神经系统的影响,除了急性期的细胞坏死,还有迟发性的神经元凋亡。目的:观察大鼠全脑缺血不同再灌注阶段海马神经元凋亡率、坏死率及凋亡调节蛋白Bcl-2和Bax的表达情况,并探讨其对全脑缺血再灌注损伤的调节作用。设计:随机对照实验。单位:首都医科大学附属北京天坛医院的神经外科和麻醉科。材料:实验于2003-01/2004-01在首都医科大学附属北京神经外科研究所完成。选择清洁级成年雄性健康Wistar大鼠33只,随机分5组,缺血再灌注24h组7只、缺血再灌注48h组7只、缺血再灌注72h组7只、缺血再灌注7d组7只和假手术对照组5只。干预:制备大鼠全脑缺血再灌注模型。分别于再灌注24,48,72h和7d取脑海马组织,用流式细胞仪检测细胞凋亡率,坏死率,Bcl-2和Bax蛋白在大鼠脑海马神经元中的表达情况。主要观察指标:①流式细胞仪检测各组大鼠脑海马组织细胞凋亡率和坏死率。②Bcl-2和Bax蛋白表达百分率。结果:33只大鼠全部进入结果分析。①缺血再灌注7d组海马神经元的凋亡率最高犤(24.59±0.97)%犦,坏死率峰值出现在缺血再灌注24h组犤(16.67±1.04)%犦,明显高于假手术对照组犤(1.28±0.50)%,(0.90±0.38)%犦(P<0.01)。②假手术对照组Bcl-2表达极低犤(1.07±0.27)%犦,但Bax有高表达犤(46.09±5.37)%犦。③Bcl-2蛋白峰值出现在缺血再灌注后48h犤(14.41±0.67)%犦,而Bax蛋白峰值出现在缺血再灌注之后72h犤(77.38±1.52)%犦。结论:全脑缺血再灌注损伤后海马神经元凋亡率逐渐增高,凋亡调控基因Bcl-2和Bax表达异常增高,提示Bcl-2和Bax蛋白参与了全脑缺血再灌注损伤的凋亡调节。     

藻酸双酯钠对大鼠急性脑缺血再灌注后Bcl-2,Bax表达的影响

目的:探讨大鼠急性脑缺血再灌注后Bcl-2,Bax表达及藻酸双酯钠对大鼠急性脑缺血再灌注后Bcl-2,Bax表达的影响。方法:实验于2004-01/04在华北煤炭医学院形态实验室完成。96只健康雄性Wister大鼠用随机数字表法随机分成4组:脑缺血组(42只)、藻酸双酯钠治疗组(42只)、假手术组(6只)、正常对照组(6只)。前两组又分为7组:即缺血2h再灌注1,3,6,12,24,48和72h组(n=6)。采用线栓法制作大鼠大脑中动脉阻断再灌注模型,用免疫组织化法分别检测缺血组与藻酸双酯钠治疗组大鼠脑缺血2h再灌注1,3,6,12,24,48和72hBcl-2,Bax的表达水平。结果:①皮质缺血周边区Bcl-2及Bax的阳性表达随再灌注时间不同而不同,分别于缺血2h再灌注3h犤(56.50±7.87)个/高倍视野犦及再灌注24h犤(61.00±8.88)个/高倍视野犦达高峰。②与缺血组相比,藻酸双酯钠治疗组Bax的表达于再灌注后12h犤(36.67±7.03)个/高倍视野犦、24h犤(50.50±6.09)个/高倍视野犦、48h犤(36.67±6.77)个/高倍视野犦及72h犤(29.83±5.38)个/高倍视野犦均显著减少(P<0.05,P<0.01),Bcl-2的表达未见明显变化(P>0.05)。结论:藻酸双酯钠可抑制脑缺血再灌注后Bax的表达,对神经细胞具有保护作用。     

大剂量硝酸甘油对大鼠离体缺血再灌注心肌细胞凋亡和Bax及Bcl-2的影响

目的:研究硝酸甘油是否对大鼠离体缺血再灌注心肌细胞凋亡和Bcl-2/Bax水平存在直接的影响。方法:实验选用SD大鼠30只,随机分为硝酸甘油组和对照组,每组15只。制备大鼠离体心肌缺血再灌注模型(缺血30min,再灌注120min)在再灌注开始时分别给以大剂量硝酸甘油(15μg/h,以950mL/L乙醇为溶剂)或950mL/L乙醇(3μL/h)。再灌注结束后,将缺血区剪下,制备石蜡切片,采用TUNEL技术检测心肌凋亡,免疫组化技术检测心肌组织的Bcl-2和Bax水平。结果:硝酸甘油组细胞发生凋亡数犤(35.2±6.7)%犦明显多于对照组细胞犤(5.2±0.8)%犦(t=28.1,P<0.01)。对照组Bcl-2表达(5.7±1.3)明显强于硝酸甘油组(1.8±0.8)(t=11.9,P<0.01)。对照组Bax表达(5.9±1.3)明显弱于硝酸甘油组(9.1±1.3)(t=9.2,P<0.01)。结论:大剂量的硝酸甘油促进了大鼠离体缺血/再灌注心肌细胞凋亡,提高了Bax蛋白的水平,降低了Bcl-2蛋白水平;大剂量硝酸甘油对Bcl-2/Bax的调节可能是其促凋亡的主要机制之一。     

地塞米松对大鼠局灶性脑缺血再灌注损伤模型TNF-α表达的干预

目的研究地塞米松对大鼠局灶性脑缺血再灌注损伤模型肿瘤坏死因子(TNF-α)表达的影响。方法大脑中动脉线栓法复制局灶性脑缺血再灌注模型。实验随机分为3组:(1)正常假手术组。(2)生理盐水组。(3)地塞米松组犤0.5mg/(kg·d)犦。生理盐水组与地塞米松组分别于缺血2h后再灌6,12,24,48h。采用HE、免疫组化及酶联免疫吸附实验(ELISA)等方法观察地塞米松对缺血再灌性脑损伤脑组织TNF-α表达的影响。结果脑缺血再灌注性损伤后TNF-α阳性细胞数,生理盐水组和地塞米松组各时间点明显多于假手术组(3.0±7.2)(q=9.38～17.98,P<0.01),再灌注6,12h,地塞米松组(45.1±5.4,81.4±17.4)明显多于生理盐水组(34.2±7.2,60.4±15.4)(q=4.67,P<0.01;q=3.50,P<0.05)。结论脑缺血再灌注后TNF-α表达的增强可能是地塞米松加重缺血再灌注性脑损伤的机制之一。     

雌激素、灯盏花对去势大鼠脑缺血损伤保护作用的叠加实验

目的:探讨去势大鼠局灶脑缺血再灌注后雌激素、灯盏花的叠加保护作用。方法:实验于1998-06/1999-04在华西医科大学的相关实验室进行。雌性SD大鼠进行双侧卵巢切除后2周再采用线栓法制备去势大鼠大脑中动脉缺血(2h)再灌注(70h)模型。50只SD大鼠随机分为雌激素组,灯盏花组、雌激素加灯盏花组、对照组和假手术组。再灌注2h和70h后分别进行神经功能缺损评分。再灌注70h显微镜下观察脑损伤区组织病理学、脑梗死体积比和脑水肿体积,测定血液中一氧化氮、白细胞介素1(IL-1)及肿瘤坏死因子(TNF)水平的变化,采用免疫组织化学方法观察脑内雌激素受体的表达。结果:再灌注70h后,与对照组神经功能缺损评分犤(1.7±0.6)分犦相比,雌激素组犤(0.3±0.5)分犦、灯盏花组犤(0.9±0.6)分犦、雌激素加灯盏花组犤(0.2±0.4)分犦明显降低(F=13.488,P<0.05);与对照组脑梗死体积比犤(31.33±1.26)%犦相比,雌激素组犤(12.52±1.40)%犦、灯盏花组犤(18.35±1.10)%犦、雌激素加灯盏花组犤(11.52±0.69)%犦明显降低(F=612.876,P<0.01);与对照组脑水肿体积犤(69.77±3.40)mm3犦相比,雌激素组犤(32.59±2.12)mm3犦、灯盏花组((51.2±4.37)mm3)、雌激素加灯盏花组犤(30.97±2.13)mm3犦明显降低(F=334.867,P<0.01);与对照组IL-1水平犤(0.9     

胰岛素对缺血再灌注心肌超微结构和细胞凋亡的影响

目的:探讨极化液(胰岛素)对家兔心肌缺血再灌注损伤的保护作用。方法:选择健康雄性家兔48只,按随机抽签法分为3组:对照组,胰岛素组,葡萄糖-钾-胰岛素(glucose-insulin-potassium,GIK)组,每组16只。制备兔心肌缺血再灌注损伤模型,分别用GIK、胰岛素、生理盐水干预分组。再灌注结束后检测心肌梗死面积,在电镜下观察心肌细胞的超微结构,原位末端标记(TUNEL)法测定凋亡心肌细胞,免疫组化SP法测定原癌基因蛋白(Bcl-2),Fas的含量。结果:与对照组比较,GIK及胰岛素组家兔心肌细胞超微结构表现为损伤减轻,心肌梗死面积明显减少犤GIK组(38.9±4.33)%,胰岛素组(40.8±5.22)%,对照组(54.42±4.27)%犦(q=4.32,3.95,P<0.05),凋亡指数犤GIK组(1.38±0.82),胰岛素组(1.28±0.54),对照组(2.15±0.19)犦(q=0.65,0.72,P<0.05)和Fas含量显著减少(q=0.07,0.06,P<0.05),Bcl-2含量增加(q=0.14,0.11,P<0.05)。GIK组和胰岛素组心肌细胞凋亡指数及Fas,Bcl-2蛋白含量比较,差异无显著性意义(P>0.05)。结论:GIK具有抗缺血再灌注损伤的作用,其机制可能是通过胰岛素调节Bcl-2和Fas介导的心肌缺血再灌注细胞凋亡而实现。     

亚低温对缺血再灌注大鼠海马CA1区Bcl-2和Bax表达动态变化的影响

目的:研究脑缺血再灌注后全身亚低温的脑保护作用及其对Bcl-2和Bax表达的动态影响,探讨亚低温脑保护作用的机制。方法:实验于2002-01/12在江苏省麻醉医学研究所(省级重点实验室)完成。实验动物选择126只SD雄性大鼠随机分为假手术组、常温缺血组和亚低温缺血组。采用Pulsinelli四血管阻断法制作大鼠全脑缺血再灌注模型,即刻全身亚低温4h,分别在再灌注后4,8,24,48,72,96h,7d,7个时间点取脑标本,进行Bcl-2和Bax免疫组织化学及苏木精-伊红染色。结果:与常温组相比,全身亚低温组海马CA1区死亡细胞数明显减少[常温比亚低温24h:(195±18)比(214±20)个/mm,P<0.05;48h:(185±17)比(215±21)个/mm,P<0.01;72h:(130±20)比(200±17)个/mm,P<0.01)];Bcl-2蛋白免疫反应强度峰值增高,持续时间延长[灰度值:常温比低温:24h(40±8比57±8,P<0.01);48h(42±6比55±8,P<0.01);72h(38±4比41±6,P<0.01)]。Bax蛋白免疫反应强度峰值减低,持续时间缩短[灰度值:常温比低温24h(32±4比24±5,P<0.05);48h(30±7比24±6,P<0.05);72h(26±4比22±5,P<0.05)]。结论:全身亚低温对缺血性锥体细胞损害有较好的保护作用。可增强具有抗凋亡的Bcl-2蛋白的表达,延长其表达持续时间,而减弱具有促凋亡的Bax表达,同时缩短其表达持续时间,此可     

亚低温状态对大鼠脑缺血再灌注损伤的保护途径

目的:大鼠局灶性脑缺血再灌注造成的脑损伤系炎症反应的细胞因子细胞间黏附分子1表达上调,并使中性粒细胞聚集的结果。髓过氧化物酶活性可作为中性粒细胞浸润的定量指标,观察亚低温状态下细胞因子和酶动态变化的特点。方法:实验于2004-09/12在武汉大学人民医院实验中心完成。选取90只SD大鼠,随机分为3组:①假手术组(n=10):仅分离并牵拉左侧颈内动脉,不阻断其血流,肛温稳定在37℃,术后24h麻醉状态下处死。②常温组(n=40):采用大鼠大脑中动脉线栓闭塞/再通法建立大鼠局灶性缺血再灌注模型,缺血期肛温稳定在37℃,分别于脑缺血3h再灌注6,24,48和72h麻醉状态下处死,每个时间点10只。③亚低温组(n=40):同前造模,缺血期间肛温保持在32～33℃,缺血结束后立即自然复温,处死时间和只数同常温组。细胞间黏附分子1阳性微血管数用免疫组化测定,同时检测髓过氧化物酶活性。结果:经补充后90只大鼠进入结果分析。①细胞间黏附分子1阳性微血管数:常温组再灌注6h缺血侧脑组织中可见到和增高,48h达到高峰犤(98.01±9.00)条犦,再灌注72h仍保持一定水平的表达,亚低温组各时间点均低于相应的常温组(P<0.05,P<0.01),但两组均高于假手术组犤(5.35±3.07)条,P<0.05,P<0.01犦。②髓过氧化物酶活性:常温组再灌注6h增高,48h达高峰,显著高于假手术组犤(0.5594±0.1662),(1.9099±0.2507),(0.2037±0.0637)kat/g,P<0.01犦,亚低温组各时间点均低于相应的常温组(P<0.05,P<0.01),但仍高于假手术组。结论:脑缺血再灌注后炎症反应是造成脑损伤的重要因素之一。亚低温所起到的脑保护作用,其途径为抑制脑缺血再灌注后缺血区细胞间黏附分子1的表达和脑组织中的髓过氧化物酶活性,从而使白细胞聚集浸润减少。     

Measurement of L-carnitine and acylcarnitines in body fluids and tissues in children and in adults

We have developed a reliable and validated radio-enzymatic method for the assay of L-carnitine and acylcarnitines, using a modification of existing methods. The sensitivity of the assay is 10 mumol/l using 10 microliters of plasma or urine. It is also suitable for measurements of carnitine in a 10 mg sample of liver or muscle obtained by percutaneous biopsy. The use of N-ethylmaleimide in the reaction mixture together with an excess of [1-14C]acetyl CoA ensures that the reaction proceeds to completion and a linear response is obtained. Using this method control ranges have been established for plasma and urine carnitine concentrations in healthy children and adults, and for the carnitine content of liver and muscle in adults. No significant difference was found between fasting and post-prandial plasma carnitine levels. An age-related increase was found in urinary total carnitine and acylcarnitine concentration throughout childhood. These data provide a reliable basis for studies of patients with abnormal carnitine and acylcarnitine metabolism, distribution and excretion.     

Activity of amikacin and ampicillin in succession and in combination

One strain each of Escherichia coli and Streptococcus faecalis were exposed to amikacin and ampicillin in combination as well as in succession. Exposure to ampicillin for 1 hr followed by amikacin for 3 or 4 hr had the greatest antibacterial activity when the antibiotics were applied in succession. The least effective exposures for both organisms were 1 hr to amikacin followed by 3 or 4 hr to ampicillin. Exposure to the antibiotics in combination each at 1 MIC had the overall greatest antibacterial activity. Simultaneous exposure to the antibiotic combination does not necessarily mean simultaneous activity of both ampicillin and amikacin on the E. coli. The cell wall autolytic activities produced by ampicillin are triggered within 10 min after physical contact with the bacteria. In contrast, amikacin requires at least 30 min after physical contact to manifest its activity on the ribosome. Although physical exposure to both antibiotics in the combination is simultaneous, the specific activity of each is in fact sequential, with ampicillin acting first. This explains the synergistic effect of the combination. It appears, therefore, that the synergistic or antagonistic affect of a drug combination is determined by the sequence and timing of the antibacterial manifestations of its components.     

Sites of in vivo extraction and interconversion of testosterone and androstenedione in dogs

The interconversion and extraction of testosterone and androstenedione across and within different tissues or areas have been studied by the constant infusion technique. The results were calculated using the (3)H/(14)C ratios and radioactive concentrations of testosterone and androstenedione obtained from afferent and efferent blood and tissues at equilibrium. In each tissue studied, the interconversion between testosterone and androstenedione inside the tissue was significantly higher than the corresponding interconversion across the tissue. The pulmonary contribution to the total interconversion between testosterone and androstenedione was far more important than that of any of the other tissues studied. The hepatic metabolic clearance rates of testosterone and androstenedione were not different from their metabolic clearance rates in the mesenteric area. The extraction of each of these compounds, although not negligible, was lower in the kidney and the femoral bed compared with the extraction in the liver and the mesenteric area. Finally, with the possible exception of the liver, testosterone and androstenedione were more completely metabolized when they originated from the cells than from afferent blood.The evaluation of these different tissue transfer constants provides more precise information concerning the relative importance of different sites in the metabolism of these interconverting hormones.     

纤维支气管镜在儿童咯血诊断与治疗中的应用

目的 评价纤维支气管镜术在儿童咯血病因诊断及治疗中的价值以及安全性.方法 应用用日本产Olympus BF 3c-40纤维支气管镜(最小外径3.6 mm)给58名咯血原因不明的患者行纤维支气管镜检查,并予镜下局部止血治疗.判断出血部位、观察病变情况和出血的原因、临床表现、其他辅助检查、治疗及转归等进行综合分析.结果 引起咯血的主要疾病为气管支气管、肺部的炎症24例(41.3%)、支气管内膜结核12例(20.7%)、支气管异物8例(13.7%)、特发性肺含铁血黄素沉着症7例(12.1%)、支气管扩张4例(6.9%)、心肺血管发育异常1例,原因不明2例.诊断阳性率为96.5%.镜下发现有活动性出血18例,镜下局部止血治疗后显效者10例,有效者8例,有效率为100%.术中并发短暂低氧血症(SaO2＜85%,＜20 s)15例,加大吸氧流量后均改善;术后发热3例均为低热,24 h后热退.结论 纤维支气管镜检查可明确出血部位及原因并可进行局部治疗,且安全的有效.     

Role of oxidants and antioxidants in atherosclerosis: results of in vitro and in vivo investigations

Both in vitro and in vivo studies have shown that oxidants are central in the development of atherosclerosis. Consequently, additional studies evaluated the protective effects of various natural and synthetic antioxidants, alone and in combination, with most studies focusing on alpha-tocopherol (vitamin E). Here, we summarize the role of oxidants in the pathomechanism of atherosclerosis. We also discuss epidemiological studies and others focused on the protective effect of vitamin E against atherosclerosis. Other antioxidants are also considered if they were included in studies involving vitamin E. The protective effect of antioxidants on atherosclerotic pathomechanisms has been confirmed in vitro, but only in some animal studies. Various epidemiological and observational studies have produced conflicting results on the protective effect of antioxidants. Most studies of primary or secondary prevention failed to show a protective effect. These conflicting results are biased by a number of factors, including differences between the study groups. Therefore, we describe these studies in detail.     

Pharmacokinetics and tolerance of flunitrazepam in neonates and in infants.

OBJECTIVE: To study the pharmacokinetics of flunitrazepam (used for sedation in neonates and infants), to determine the influence of both gestational and postnatal age on the pharmacokinetic parameters, and to analyze the relationship between the hemodynamic parameters and flunitrazepam plasma concentration. METHODS: Flunitrazepam was infused for 20 minutes as a single dose (0.2 mg x kg(-1)) and as multiple doses (0.1 mg x kg(-1)). Six to eight 1-mL blood samples were collected per patient. Flunitrazepam plasma concentration was measured by gas chromatography-mass spectrometry. RESULTS: Thirty-one patients (25 neonates and six infants) were included in the study. Only three of them received multiple doses. After the single dose (n = 28), half-life was 22.6 +/- 7.3 hours, clearance was 0.15 +/- 0.14 L x kg x h(-1), and volume of distribution was 4.6 +/- 4.1 L x kg(-1) (mean +/- SD). Plasma clearance and volume of distribution significantly increased with postnatal age (P < .05), but no pharmacokinetic parameter varied significantly with gestational age. Diastolic blood pressure significantly decreased with increasing flunitrazepam plasma concentrations (P < .05). CONCLUSION: Postnatal age but not gestational age influenced flunitrazepam pharmacokinetic parameters in neonates and infants. Diastolic blood pressure was inversely correlated to flunitrazepam plasma concentration.