细胞外信号调节激酶在缺糖缺氧/复糖复氧神经元中的表达与银杏叶提取物的调节作用

目的研究细胞外信号调节激酶(ERK1/2)在缺糖缺氧/复糖复氧神经元中的表达以及银杏叶提取物的调节作用。方法利用原代培养的皮层神经元,通过去除培养液中的糖和氧(oxygen and glucose deprivation,OGD)模拟缺血缺氧,恢复糖氧供给模拟再灌注。通过免疫蛋白印迹法测定ERK1/2、p-ERK1/2蛋白的表达。再灌注时加用银杏叶提取物(EGB761)观察其对神经元保护作用以及时ERK1/2表达的调节作用。结果缺糖缺氧/复糖复氧后神经元p-ERK1/2表达明显下降,EGB761可剂量依赖地保护神经元活性,并可上调p-ERK1/2蛋白的表达。结论EGB761对培养的神经元缺糖缺氧/复糖复氧损伤有保护作用,该作用可能与激活ERK信号转导通路有关。     

大鼠海马神经元体外氧糖剥夺/复氧模型的构建

目的 建市体外培养的大鼠海马神经元氧糖剥夺(OGD)/复氧实验模型,并尝试确定该模型最合适的缺氧缺糖时间点.方法 新生SD乳鼠海马神经元原代培养7 d后,随机(随机数字法)分为OGD组和对照组.OCD组根据氧糖剥夺时间的不同又分为1 h,2 h,4 h,6 h,8 h,10 h亚组.OGD组细胞置于含0.5%氧气的三气培养箱,同时将培养液换成无糖Earle氏液,模拟体内脑缺血损伤.复氧复糖24 h后观察对照组和OCD各组的神经元形态,测定MTT细胞光密度值(OD)和培养液LDH含量,流式细胞仪检测细胞凋亡率.所得数据采用SPSS 16.0统计软件行单因素方差分析(Dunnett-t检验)和Spearman等级相关分析.结果 随着缺氧缺糖时间的延长,OGD各组神经元形态学损伤逐步加重,细胞OD值和存活率逐渐下降(rs=-0.961和rs=-0.966,P<0.01),LDH值逐渐升高(rs=0.990,P<0.01),细胞凋亡率明显增加,与对照组比较差异均有统计学意义(P<0.05).OGD6 h时,细胞的凋亡率接近50%.结论 成功建立了大鼠海马神经元体外氧糖剥夺/复氧模型,结合形态学改变和细胞凋亡率,建议将6 h作为该模型合适的缺氧缺糖损伤时间.     

缺氧损伤对新生大鼠海马神经干细胞细胞活力及分化潜能的影响

[目的]通过建立缺氧损伤模型,观察缺氧损伤对神经干细胞活力及分化潜能的影响.[方法]采用机械吹打、无血清悬浮培养,95%N2、5%CO2比率的缺氧气体体外分离培养新生大鼠海马神经干细胞,建立缺氧模型,免疫细胞化学鉴定神经干细胞及其分化后的细胞类型,MTT、LDH等方法比较缺氧培养后神经干细胞的变化,并观察缺氧培养1、2、4、6、8 h后神经元分化的比率.[结果]新生大鼠海马可分离出的大量细胞Nestin染色阳性,证实为神经干细胞,分离出的神经干细胞可分化为神经元、胶质细胞等神经细胞类型;95%N2、5%CO2比率的缺氧气体培养6 h后神经干细胞MTT值由缺氧前0.368±0.104降至0.268±0.071(P＜0.05),LDH漏出量由缺氧前10.369±0.379 unit/(ml·min)增加到13.987±1.240 unit/(ml·min)(P＜0.05);神经元的分化比率稍增加,但无显著差异(P》0.05).[结论]新生大鼠海马内提取的细胞为神经干细胞,神经干细胞缺氧培养6 h后可以建立神经干细胞缺氧损伤模型,短时间缺氧培养对细胞分化的类型无明显改变.     

银杏叶提取物对缺血-再灌流神经元损伤的保护作用及其信号转导机制

目的研究银杏叶提取物(EGB761)对缺糖缺氧/复糖复氧神经元损伤的保护作用,并探讨其主要细胞内信号转导机制。方法利用原代培养的皮层神经元,通过去除培养液中的糖和氧(oxygen and glucose deprivation,OGD)模拟缺血缺氧,恢复糖氧供给模拟再灌流。通过免疫蛋白印迹法测定Akt、磷酸化Akt(p-Akt)蛋白的表达。再灌流时加用银杏叶提取物(EGB761)观察其对神经元保护作用以及对Akt、p-Akt表达的调节作用。结果缺糖缺氧/复糖复氧后神经元p-Akt表达明显下降,EGB761可剂量依赖地保护神经元活性,并可上调因缺糖缺氧/复糖复氧而降低的p- Akt蛋白的表达,但该作用不被Ly294002所阻断。结论EGB761对培养的神经元缺糖缺氧/复糖复氧损伤有保护作用,该作用可能与非PI3K依赖的p-Akt信号转导通路激活有关。     

脑溢安对新生大鼠海马神经干细胞缺氧损伤及白介素-6和白介素-6mRNA表达的影响

目的:探讨中药脑溢安对神经干细胞缺氧损伤的保护作用机制。方法:体外培养神经干细胞来自新生3—5天的SD大鼠海马组织,将神经干细胞移入厌氧培养箱(37℃、95%N2和5%CO2)内培养6h造缺氧损伤,用免疫组化及原位杂交技术研究脑溢安对缺氧损伤神经干细胞及白介素-6(IL-6)和IL-6mRNA表达的影响。结果:脑溢安能促进缺氧损伤神经干细胞存活;缺氧损伤后神经干细胞内IL-6和IL-6mRNA表达增强,脑溢安血清组IL-6及IL-6mRNA表达显著高于模型组和正常血清对照组(P<0.01)。结论:脑溢安可能通过增强IL-6和IL-6mRNA的表达发挥其对缺氧损伤神经干细胞的保护作用。     

移植骨髓间充质干细胞后的脑脊液促进神经干细胞的分化

目的观察骨髓间充质干细胞（BMSC）移植于损伤脊髓后,所提取的脑脊液（BMSCs-CSF）对体外培养的神经干细胞向神经元细胞分化的影响,探讨BMSCs促进神经干细胞分化的机制。方法培养BMSCs及神经干细胞;制作大鼠脊髓损伤模型;将体外培养的BMSCs注入脊髓损伤区,于术后3d、5W分别提取脑脊液,并注入神经干细胞培养液中,观察神经干细胞向神经元细胞的分化。试验分为五组,A：无脊髓损伤组脑脊液;B1：脊髓损伤3天后收集的脑脊液;B2：脊髓损伤组5周后收集的脑脊液;C1：脊髓损伤并行BMSCs移植,在3天后收集的脑脊液;Q2：脊髓损伤并进行BMSCs移植,在5周后收集的脑脊液。结果加入脑脊液培养72小时后在倒置显微镜下观察细胞形态结果显示A、B1、B2各组神经干细胞球无贴壁,悬浮于培养基中,神经干细胞无细胞突形成;C1组显示神经干细胞球贴于培养板底部,并有细胞突生长。C2组显示仅有少量的神经干细胞球贴壁,细胞突短小少见。结论BMSCs移植于损伤的脊髓后所提取的脑脊液（BMSCs-CSF）具有促进神经干细胞向神经元细胞分化的作用。     

异氟醚预处理对离体神经元损伤模型的作用

目的：观察异氟醚对氧糖剥夺离体神经元损伤的保护作用。方法：新生小鼠神经元原代培养14d后,造成氧糖剥夺离体神经元缺血模型,4h后复氧、复糖,模拟再灌注模型。随机分为3组：C组为对照组;A组用1MAC异氟醚通入培养液预处理1h;B组用2MAC异氟醚通入培养液预处理1h。各组分别于复氧复糖后4、12和24h取神经元细胞计数后作四唑盐（MTT）比色试验。并取复氧、复糖24h各组神经元细胞做台盼蓝染色,计阳性细胞数百分比。另取原代培养神经元,造成氧糖剥夺离体神经元缺血模型30min后分成两组后分别按C组和A组处理,复氧、复糖4h后,免疫组化SABC法染色,比较nNOS和iNOS活性。结果：复氧、复糖后A组和B组MTT各时点值均大于C组,且台盼蓝染色阳性细胞数百分比A组和B组均小于C组,但A组和B组间无显著差异。免疫组化染色结果显示：A组nNOS和iNOS的表达灰度值均低于C组。结论：异氟醚能减轻氧糖剥夺离体神经元的损伤,抑制神经元nNOS和iNOS活性是其可能机制之一。     

视网膜干细胞培养上清液诱导骨髓间充质干细胞的分化

背景：骨髓间充质干细胞向神经细胞诱导分化可为神经系统受损伤后的修复和再生带来了新的希望。
　　目的：探讨骨髓间充质干细胞在视网膜干细胞培养上清液诱导条件下向神经元细胞分化。
　　方法：采用全骨髓培养方法,用视网膜干细胞培养上清液诱导骨髓间充质干细胞,通过免疫荧光染色鉴定其分化的结果。
　　结果与结论：诱导72 h,骨髓间充质干细胞表达神经干细胞的特异性抗体巢蛋白和神经元中的标志性微管相关蛋白微管相关蛋白2。视网膜干细胞培养上清液能够促进骨髓间充质干细胞向神经元样细胞分化,提示视网膜干细胞可能分泌神经生长因子。     

大鼠局灶性脑缺血后Cdh1 mRNA水平及Cdh1蛋白分布的改变

目的:探讨大鼠局灶性脑缺血损伤后Cdh1 mRNA水平的变化及其在不同类型神经细胞中的分布变化。方法:雄性成年SD大鼠30只,线栓法建立右侧大脑中动脉缺血模型,分别于缺血前和缺血再灌注后1、3、7d采用实时荧光定量PCR技术检测缺血侧和非缺血侧脑组织中Cdh1 mRNA的水平;缺血再灌注后7d免疫荧光双标法检测Cdh1在神经元和星形胶质细胞中的分布情况。结果:脑缺血前,两侧脑组织中Cdh1 mRNA的水平差异无统计学意义。缺血再灌注后1、3、7d,缺血侧脑组织Cdh1 mRNA水平均低于非缺血侧脑组织(P0.05);缺血再灌注后7d,与非缺血侧相比,缺血侧神经元明显减少,星形胶质细胞增多;在非缺血侧Cdh1主要表达于神经元,星形胶质细胞中表达较少,在缺血侧Cdh1在神经元及激活的星形胶质细胞中均有表达。结论:大鼠局灶性脑缺血损伤后,缺血侧Cdh1 mRNA的水平较非缺血侧降低,且Cdh1在神经元和星形胶质细胞中分布情况发生变化。     

脑源性神经营养因子对缺氧神经元保护与细胞外信号调节激酶的关系

背景：有研究者发现激活细胞外信号调节激酶的上游激酶-细胞外信号调节激酶（Extracellular signal—regulated kinase，ERK）途径并没有促神经元存活的作用，还有一些研究者发现激活该信号通路不仅不能对细胞起到保护作用，反而可促进细胞死亡。 目的：实验拟观察体外培养神经元急性缺氧损伤中ERK1／2通路在脑源性神经营养因子抗缺氧损伤中的作用。 设计、时间及地点：随机对照分组设计，于2005—03／2006—04在华西医院移植工程及移植免疫实验室完成。 材料：孕17～19d的SD雌鼠，清洁级，体质量350～400g：ERK1／2特异性阻滞剂U0126购白Calbiochem公司；脑源性神经营养因子购白R＆D公司。 方法：体外培养孕17～19d的胚鼠大脑皮质神经元，7d后作缺氧培养，并随机分为5组，缺氧组：单纯神经元缺氧培养；脑源性神经营养因子组：缺氧培养前30min加入脑源性神经营养因子50μg／L；U0126组：缺氧培养前1h加入10μmol／L的U0126；U0126＋脑源性神经营养因子组：缺氧培养前1h加入10μmol／L的UO126，30min后加入50μg/L脑源性神经营养因子；基线对照组：不做缺氧培养也不加入任何物质。 主要观察指标：采用四甲基偶氮唑盐法检测0-8h内各组之间细胞存活率的差别；蛋白免疫印迹检测0-6h内各组神经元ERK1／2和磷酸化ERK1／2、cAMP反应元件结合蛋白（cAMP responsive element—binding protein，CREB）和磷酸化CREB的表达变化。 结果：①脑源性神经营养因子组神经元活性在缺氧后各时间点显著高于缺氧组（P〈0．01）；U0126＋脑源性神经营养因子组细胞活性在缺氧后各时间点显著低于脑源性神经营养因子组（P〈0．01）。②外源性脑源性神经营养因子对总ERK和总CREB的表达无影响，但可显著增加磷酸化ERK和磷酸化CREB的表达：U0126对总ERK表达无影响，但可显著抑制脑源性神经营养因子作用后磷酸化ERK的上调，同时U0126也可明显抑制脑源性神经营养因子介导的磷酸化CREB的表达。 结论：阻滞ERK1／2传递后脑源性神经营养因子对缺氧神经元的保护作用迅速降低，提示ERK1/2是体外培养神经元缺氧损伤中陆源件神绎营养因子保护作用的重要涂径。     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

俯卧位通气对高海拔地区肺复张治疗无效急性呼吸窘迫综合征患者氧合的影响

目的 探讨俯卧位通气对高海拔地区肺复张术(RM)治疗无效急性呼吸窘迫综合征(ARDS)患者的治疗作用.方法 从海拔2260m的地区医院筛选RM治疗无效的41例ARDS患者[平均氧合指数( PaO2/FiO2)较RM前升高＜20％视为RM无效],依不同病因分为肺内源性ARDS组(ARDSp组)和肺外源性ARDS组(ARDSexp组),每组再按信封法随机分为俯卧位组和仰卧位组,即ARDSp俯卧位组(11例)、ARDSp仰卧位组(9例)、ARDSexp俯卧位组(10例)、ARDSexp仰卧位组(11例).在通气前及通气1、2、3、4h监测动脉血氧分压( PaO2)、PaO2/FiO2、静态顺应性(Cst)、气道阻力(Raw)的变化.结果 通气lh时,ARDSexp俯卧位组PaO2/FiO2( mm Hg,l mm Hg=0.133 kPa)即较通气前显著升高(157.4±40.6比129.3±48.7,P＜0.05),并随通气时间延长呈持续增高趋势,4h达峰值(219.1 ±41.1);且ARDSexp俯卧位组通气3h内PaO2/FiO2较其他3组显著增高,另3组间则差异无统计学意义.ARDSp俯卧位组、ARDSexp俯卧位组通气4h时PaO2/FiO2均较相应仰卧位组显著增高(208.8±39.7比127.4±47.1,219.1±41.1比124.9±50.8,均P＜0.05).4组通气前后Cst无显著改变,各组间差异也无统计学意义.ARDSp俯卧位组通气4h时Raw(cmH2O·L-1·s-1)较通气前显著降低(6.8±1.7比10.7±1.8,P＜0.05),且明显低于其他3组;其他3组各时间点Raw组内及组间比较差异均无统计学意义.结论 俯卧位通气作为ARDS机械通气重要策略之一,可以改善RM无效高原ARDS患者的氧合,为抢救患者赢得宝贵的时间.     

Digital imaging among medical facilities

The Department of Veterans Affairs (VA) in the USA operates a network of 172 medical centres which all utilize a hospital information system (HIS) which has been developed and is currently maintained by the VA. During the past several years, an image management and communication module has been developed, installed and clinically utilized at the Washington DC and Maryland VA Medical Centres. This image management and communication system, referred to as the decentralized hospital computer program (DHCP) imaging system, is fully integrated with a commercial picture archiving and communication system (PACS). The system is utilized to capture, archive, and display all images generated within the hospital including radiology, nuclear medicine, pathology, endoscopy, bronchoscopy, and dermatology, intraoperative photographs, ECG data, and a limited number of paper documents. The ultimate goal of the project is to have all patient text and image data available at any clinical workstation to any authorized user anywhere within the network of medical centres. Clinical requirements for an imaging workstation include ease of use, rapid and reliable access to the complete set of patient information, and images which are of acceptable quality to meet the requirements of the user and the subspecialty. Patient confidentiality and data security must be safeguarded at all times. Integration of the images with the remainder of the patient's database was found to be critical to the success of the project. The experience at the Washington and Maryland facilities suggests that an imaging system that is successfully integrated with a hospital information system can provide substantial clinical and economic benefits both within and among medical centres. Clinical acceptance and utilization of the system has been excellent, particularly in diagnostic radiology where DHCP Imaging has been interfaced to a commercial PAC system. Based upon this initial experience, the VA has begun to deploy the system throughout its large network of medical centres.     

Myocardial elastography at both high temporal and spatial resolution for the detection of infarcts

Myocardial elastography is a novel method for noninvasively assessing regional myocardial function, with the advantages of high spatial and temporal resolution and high signal-to-noise ratio (SNR). In this paper, in-vivo experiments were performed in anesthetized normal and infarcted mice (one day after left anterior descending coronary artery [LAD] ligation) using a high-resolution (30 MHz) ultrasound system (Vevo 770, VisualSonics Inc., Toronto, ON, Canada). Radiofrequency (RF) signals of the left ventricle (LV) in longitudinal (long-axis) view and the associated electrocardiogram (ECG) were simultaneously acquired. Using a retrospective ECG gating technique, 2-D full field-of-view RF frames were acquired at an extremely high frame rate (8 kHz) that resulted in high-quality incremental displacement and strain estimation of the myocardium. The incremental results were further accumulated to obtain the cumulative displacements and strains. Two-dimensional and M-mode displacement images and strain images (elastograms), as well as displacement and strain profiles as a function of time, were compared between normal and infarcted mice. Incremental results clearly depicted cardiac events including LV contraction, LV relaxation and isovolumetric phases in both normal and infarcted mice, and also evidently indicated reduced motion and deformation in the infarcted myocardium. The elastograms indicated that the infarcted regions underwent thinning during systole rather than thickening, as in the normal case. The cumulative elastograms were found to have higher elastographic SNR (SNR(e)) than the incremental elastograms (e.g., 10.6 vs. 4.7 in a normal myocardium, and 6.0 vs. 2.4 in an infarcted myocardium). Finally, preliminary statistical results from nine normal (m = 9) and seven infarcted (n = 7) mice indicated the capability of the cumulative strain in differentiating infracted from normal myocardia. In conclusion, myocardial elastography could provide regional strain information at simultaneously high temporal (>/=0.125 ms) and spatial ( approximately 55 microm) resolution as well as high precision ( approximately 0.05 microm displacement). This technique was thus capable of accurately characterizing normal myocardial function throughout an entire cardiac cycle, at the same high resolution, and detecting and localizing myocardial infarction in vivo.     

手转胎头术失败的原因与分娩结局临床分析

目的 探讨手转胎头术失败的原因与分娩结局.方法 选择2008年1月至2010年12月于我院住院分娩的持续性枕横位、枕后位产妇198例,根据行手转胎头术后结果分为成功组126例、失败组72例.比较两组分娩结局,对比分析失败原因.结果 失败组胎儿体质量≥3500 g的发生率[76.4％(55/72)]明显高于成功组[31.7％(40/126)],差异有统计学意义(x2=30.177,P=0.001)、失败组宫缩乏力发生率[58.3％(42/72)]高于成功组[38.1％ (48/126)],差异有统计学意义(x2=7.569,P=0.006)、失败组骨盆临界或轻度狭窄发生率[38.9％ (28/72)]高于成功组[23.8％(30/126)],差异有统计学意义(x2 =5.030,P=0.002)、失败组手转胎头时机不当(宫口开大＜6 cm、胎头位于坐骨棘上及宫口开大8～10 cm、胎头位于坐骨棘下≥2 cm)发生率[61.1％(44/72)]高于成功组[38.9％(49/126)],差异有统计学意义(x2=9.084,P=0.003).失败组母儿并发症(产后出血、产褥病率、胎儿窘迫、新生儿窒息)发生率高于成功组(x2 =9.586,P=0.002、x2=9.334,P=0.002、x2=5.910,P=0.015、x2=5.240,P=0.022)、失败组剖宫产发生率[72.2％(52/72)]明显高于成功组[34.1 ％(43/126),x2=26.641,P=0.001)].结论 手转胎头术能使难产变顺产,降低剖宫产率,减少母儿并发症,但须积极预防、处理导致手转胎头术失败的原因,对矫正失败后继续矫正及试产应慎重.     

Clinical Pharmacokinetics of Morphine

Morphine, the most widely used mu-opioid analgesic for acute and chronic pain, is the standard against which new analgesics are measured. A thorough understanding of the pharmacokinetics of morphine is required in order to safely and effectively use this analgesic in a wide variety of patients with different levels of organ function. A MEDLINE search was conducted to identify literature published between 1966 and January 2002 relevant to the pharmacokinetics of morphine. These publications were reviewed and the literature summarized regarding unique and clinically important elements of morphine disposition relative to its parenteral administration (including intravenous, intramuscular, subcutaneous, epidural and intrathecal administration), absorption profile (immediate release, controlled release, and sublingual/buccal, and rectal administration), distribution, and its metabolism/ excretion. Special populations, including infants, elderly, and those with renal/liver failure, have a unique morphine pharmacokinetic profile that must be taken into account in order to maximize analgesic efficacy and reduce the risk of adverse events.     

Evidence-Based Pain Management and Palliative Care in Issue Four for 2009 of The Cochrane Library

ABSTRACT

The Cochrane Library of Systematic Reviews is published quarterly. Issue 4 for 2009 contains 4027 complete reviews, 1906 protocols for reviews in production, and 11447 one-page summaries of systematic reviews published in the general medical literature. In addition, there are citations of 600,000 randomized controlled trials, and 12,200 cited papers in the Cochrane methodology register. The health technology assessment database contains over 7500 citations. This edition of the Library contains 90 new reviews, of which 19 have potential relevance for practitioners in pain and palliative medicine.     

Wie zuverlässig sind Angaben zu nichtentzündlichen ausgedehnten Schmerzen?

ZusammenfassungFragestellung Es wurde geprüft, wie sich der Differenziertheitsgrad zweier Schmerzmessmethoden auf Angaben zur Ausgedehntheit klinischer Schmerzen auswirkt. Zugleich wurde der Referenzzeitraum variiert, über den die Patienten berichten sollten.Methode Erfasst wurde der Einfluss zu Lasten der Befragungsdifferenziertheit durch den Vergleich zweier Körperschema-Bildvorlagen. Drei Referenzzeiträume (Schmerz aktuell, letzte Woche, letztes halbes Jahr) wurden vorgegeben.Ergebnisse Patienten mit ausgedehnten Schmerzen gaben bei differenzierter Befragung um so mehr Schmerzen an, je weiter die Schmerzen zurück lagen und je größer der Berichtszeitraum war. Patienten mit gelenknahen Schmerzen gaben bei hoch differenzierter Befragung weniger ausgedehnte Schmerzen in der Vergangenheit an als bei globaler Einschätzung. Patienten mit Rückenschmerzen berichteten bei differenzierter Befragung zum aktuellen Schmerz über weniger ausgedehnte Schmerzen als bei globaler Befragung.Schlussfolgerung Die Angaben zur Schmerzausdehnung variieren vor allem bei Patienten mit ausgedehnten Schmerzen in Abhängigkeit von der Differenziertheit der Befragung. In diesen Fällen ist die Wahrscheinlichkeit erhöht, dass sich die Beschwerdesymptomatik zumindest teilweise erst in der Reaktion auf die situativen Befragungsbedingungen konstituiert und daher nicht auf andere Befragungsbedingungen generalisiert werden kann.