人脐静脉内皮细胞分离培养及鉴定技术

目的探讨人脐静脉内皮细胞(HUVEC)体外分离原代培养的方法,并总结对培养的细胞鉴定方法。方法通过胰酶灌注法从人脐带获取内皮细胞进行分离培养,并采用免疫组化法及光镜和透射电镜观察超微结构鉴定所获得的细胞系。结果分离的HUVEC在体外7-10天左右可长成单层,光镜下胞体为单层铺路石状排列。第VⅢ因子相关抗原的检测为阳性。透射电镜下观察培养的内皮细胞胞浆内可见Weibel-Palade小体。结论用胰酶灌注法是获得脐静脉内皮细胞的有效方法。本方法为血管内皮细胞的研究提供了实验模型。     

人脐静脉内皮胞细体外培养及鉴定

目的探索人脐静脉内皮细胞体外培养及鉴定的方法。方法采用0.1%Ⅰ型胶原酶分离脐静脉内皮细胞,加入含10%胎牛血清及人AB血清的M1640培养基,在37℃,5%CO2孵箱中培养,以免疫组化方法对内皮细胞进行鉴定。结果原代培养细胞在接种4 h后开始贴壁生长,5～7 d后融合成单层,倒置相差显微镜下观察细胞呈鹅卵石状排列,有接触抑制现象,免疫组化显示细胞胞浆中人Ⅷ因子相关抗原阳性。结论用胶原酶灌注消化脐静脉是获得内皮细胞的一种可靠的方法,有助于体外研究血管内皮细胞模型的构建。     

人滑膜细胞的分离培养与鉴定

[目的]建立人滑膜细胞体外分离培养的方法，为进一步的实验研究奠定基础。[方法]分别用胶原酶消化法及组织块培养法分离和培养人滑膜细胞，并通过形态学和免疫组化进行鉴定。[结果]培养的滑膜细胞具有成纤维细胞的形态和特征；免疫组化染色显示Vimentin阳性，CD68阴性，滑膜细胞呈长梭形极向生长。[结论]两种方法均成功的培养出了人滑膜细胞，适合做为实验研究的靶细胞。     

大鼠胚胎肝干细胞的分离方法比较

目的比较两种体外分离培养原代大鼠胚胎肝干细胞(EHSCs)方法,并对其进行鉴定。方法用胶原酶灌注法及剪碎消化法分离不同年龄的大鼠胚胎肝干细胞,用含10%优等胎牛血清的H-DMEM培养液培养。利用免疫细胞化学方法在激光共聚焦扫描显微镜下观察该细胞表面抗原的表达情况,进行鉴定。结果两种方法分离的大鼠胚胎肝干细胞体外培养24 h贴壁,5 d左右可长成单层,光镜下为致密圆形细胞,边缘清楚。8 d后细胞铺展,呈上皮样。表达甲胎蛋白(AFP)抗原、细胞角蛋白(CK)18、19。结论所分离的细胞经鉴定为大鼠胚胎肝干细胞,可在体外培养条件下迅速扩增,两种方法所分离培养的肝细胞形态基本一致。     

乳兔成骨细胞原代培养与鉴定：改良胶原酶与胰酶的分段消化

背景：成骨细胞获取的方法较多,如何简便而迅速的获得高纯度的成骨细胞成为研究的热点。目的：比较组织块法、胶原酶消化法、改良胶原酶和胰酶分段消化法体外培养纯化乳兔颅骨来源的成骨细胞结果及其细胞的生物学特点。方法：取新生24 h内新西兰大白兔乳兔颅盖骨,采用组织块法、胶原酶消化法和改良胶原酶和胰酶分段消化法分离获取兔原代成骨细胞,并进行传代培养。通过倒置显微镜下形态学观察、锥虫蓝排斥法计数活细胞率及MTT法绘制细胞生长曲线、茜素红染色、细胞培养上清液碱性磷酸酶和骨钙素检测、Ⅰ型胶原和Ⅲ型胶原免疫组织化学法和RT-PCR检测骨钙素和Ⅰ型胶原mRNA的表达等对成骨细胞进行鉴定。结果与结论：分离培养的成骨细胞均一性好、增殖能力强,具备成骨细胞的典型特征,茜素红染色阳性,Ⅰ型胶原免疫组织化学染色阳性,Ⅲ型胶原免疫组织化学染色阴性,细胞培养上清液碱性磷酸酶、骨钙素均有表达,PT-PCR结果有Ⅰ型胶原蛋白和骨钙素mRNA表达。改良胶原酶和胰酶分段消化法较传统胶原酶消化法有更高的细胞获取率,更好的细胞活性,且比传统胶原酶消化法耗时短（P〈0.05）;组织块法操作方法最为简单,细胞活性最高,但是细胞产出率最低、耗时最长,不适合用于成骨细胞大规模培养。用改良的胶原酶和胰酶分段消化法可获得数量较多且纯度较高的成骨细胞,可以成为一种相对可靠、有效的原代成骨细胞的体外培养方法。     

人颌骨骨膜成骨细胞的体外分离与培养

目的探讨人下颌骨骨膜分离培养成骨细胞的可行性。方法切取人健康的下颌骨骨膜,用胰酶、胶原酶分步消化法分离出成骨细胞,利用差异贴附原理对分离出的细胞进行纯化,对成骨细胞进行体外培养与扩增,倒置显微镜下观察细胞的生长、增殖与黏附情况,利用碱性磷酸酶染色和免疫组织化学方法检测人成骨细胞Ⅰ型胶原表达来对成骨细胞进行鉴定。结果人下颌骨骨膜成功分离出成骨细胞并进行体外培养与扩增,免疫组化学方法检测出Ⅰ型胶原表达,碱性磷酸酶染色阳性反应率达95％以上。结论自人下颌骨骨膜分离培养成骨细胞的方法切实可行。     

成年兔关节软骨创伤模型中软骨细胞的分离及体外培养观察

目的观察成年兔关节软骨损伤模型中能获得的关节软骨和软骨细胞数量,探讨不同浓度的Ⅱ型胶原酶在不同时间段分离软骨细胞的能力及体外培养所获得的软骨细胞的生物学活性。方法建立成年兔关节软骨损伤模型,用0.1%~0.2%Ⅱ型胶原酶消化4小时,每小时收集消化下来的兔膝关节软骨细胞,计数细胞收获率和细胞存活率。体外培养原代及传代细胞,观察细胞形态和生长规律,绘制生长曲线,检测细胞合成Ⅰ、Ⅱ型胶原、蛋白多糖能力。结果成年兔膝关节软骨细胞的收获率平均为3603.6×104个/克,细胞存活率平均为97.3%,胶原酶浓度与细胞的获取率无显著关系,但与细胞的存活率相关,在消化时间超过3h后细胞的存活率显著降低,在培养过程中出现易老化现象,原代细胞贴壁时间为48~72h,甲苯胺蓝异染反应较强,Ⅱ型胶原免疫组化染色反应呈强阳性,Ⅰ型胶原免疫组化染色为阴性,传3代细胞形态由卵圆形、多角形变为梭形,甲苯胺蓝染色异染反应较弱,免疫组化Ⅰ型胶原为强阳性而Ⅱ型胶原表达极弱。结论0.1~0.2%Ⅱ型胶原酶消化、分批收取细胞具有较高的细胞收获率和存活率,体外培养时,从创伤后关节软骨中获得的原代细胞和1代细胞具有良好的软骨表型,第3代及以后的细胞生物学活性低下。     

II型胶原酶消化结合组织块贴壁法分离培养兔髓核细胞

背景：椎间盘为负重却缺乏血运的组织,在体外培养过程中存在表型丢失问题,因而容易发生退行性改变,但椎间盘退变的机制尚不明确。 目的：探讨兔髓核细胞分离、贴壁培养、扩增和鉴定的方法,并观察髓核细胞在不同代次的生长特性。 方法：应用Ⅱ型胶原酶消化法和组织块贴壁法相结合的方法,分离、纯化髓核细胞并进行体外扩增,用倒置显微镜观察各代细胞的形态、生长状况,计数细胞数量,并绘制细胞生长曲线。苏木精-伊红染色后光镜下观察细胞形态,免疫细胞化学方法检测细胞Ⅱ型胶原和聚集蛋白聚糖的表达情况,并进行细胞鉴定。 结果与结论：成功的实现了兔髓核细胞体外分离、培养及扩增。生长特性观察发现,髓核细胞第1-3代细胞增殖能力强,活力旺盛,但随着传代次数的增加,细胞增殖能力程逐渐下降的趋势。分离培养的髓核细胞阳性表达聚集蛋白聚糖和Ⅱ型胶原。体外采用Ⅱ型胶原酶消化法和组织块贴壁法相结合,可获得高纯度的髓核细胞,培养的髓核细胞呈类圆形或多角形生长,第1-3代细胞生长活性较强。     

改进酶消化法培养SD大鼠成骨细胞及其鉴定

摘要 目的：改进SD大鼠成骨细胞的体外分离、培养方法并进行功能鉴定。方法：将新生SD大鼠处死，无菌条件下取出颅骨，剔净骨膜后剪成1mm3大小组织块。组织块经0.25%胰蛋白酶消化20min，继以0.1% II型胶原酶消化60 min，收集上清离心，所得成骨细胞接种于培养瓶中并行“多次贴壁法”纯化。观察细胞形态学，选用ALP Gomori 钙钴法染色，钙结节茜素红法染色及I型胶原免疫组化染色等方法进行鉴定。 结果：培养的细胞具有典型的成骨细胞形态特征，在体外培养时可维持其在体内的功能:合成碱性磷酸酶、形成矿化结节，I型胶原染色阳性。结论：用改进后的酶消化法分离、培养SD大鼠成骨细胞，更能减少胰蛋白酶在消化过程中对细胞造成的损伤，所获成骨细胞量多、纯度高，操作简易可行，可作为骨组织工程种子细胞常规的培养方法。     

大鼠髁突软骨细胞培养及生物学特性研究

目的 探讨大鼠颞下颌关节髁突软骨细胞的培养方法,观察细胞的生物学特性。方法 分离3只Wistar大鼠髁突软骨,采用Ⅱ型胶原酶消化法联合组织块法获取髁突软骨干细胞,采用MTT法检测细胞生长曲线,采用甲苯胺蓝染色、Ⅱ型胶原免疫组织化学法和Ⅱ型胶原免疫荧光法鉴定细胞来源。结果 Ⅱ型胶原酶消化法联合组织块法消化髁突软骨组织18~20h可获得最大量细胞;体外培养软骨细胞主要呈多角形,第1~4代细胞具有稳定生物学特性,5代及5代以后逐渐出现老化细胞;MTT检测结果显示第3代细胞可最快达指数生长期;髁突软骨细胞经甲苯胺蓝染色、Ⅱ型胶原免疫组织化学染色及免疫荧光染色鉴定,结果均为阳性表达,细胞来源于软骨。结论 Ⅱ型胶原酶消化法联合组织块法可作为髁突软骨细胞获取方法;4代以内细胞具有稳定的生物学特性,可用于其他实验。     

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.     

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.     

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.     

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.