改良分步消化法培养原代软骨细胞

背景：胰蛋白酶和细菌胶原酶结合使用消化关节软骨基质获得大量纯度高的软骨细胞的方法步骤繁琐、过程复杂,容易污染,但更好的简单易行、安全可靠的方法至今少有报道。目的：采用改良分步消化法进行软骨细胞培养,以获取大量纯净的软骨细胞。方法：将新西兰白兔6只随机分2组,酶消化法组运用酶消化法分两步获取原代软骨细胞,对照组用传统法进行原代软骨细胞培养。培养1周后观察两组培养的软骨细胞的生长状态,并进行细胞鉴定、计数,评估改良后的方法对细胞的影响。结果与结论：酶消化法组采用0.2%Ⅱ型胶原酶消化软骨细胞,与对照组相比,可将6h以上的消化时间缩短至3h。两组原代软骨细胞培养24h均多呈圆形,悬浮状态,48h后贴壁,培养1周后,两组软骨细胞可铺满培养瓶底。结果证实,采用改良分步消化法进行软骨细胞培养,在缩短了消化时间的同时其细胞生长及形态变化均无改变,可以顺利获取大量纯净的软骨细胞。     

骨关节炎软骨细胞的体外分离培养

背景:由于体外分离培养骨关节炎患者软骨细胞存在难度,同时软骨组织中因富含大量的基质,且软骨细胞分布于其中的软骨陷窝中,因此与其他细胞相比,软骨细胞的分离更为困难.目的:从骨关节炎行人工膝关节置换者的关节软骨中分离软骨细胞,并改良其酶消化法分离软骨组织,观察软骨细胞的生物学特性.设计、时间及地点:对比观察,实验于2007-04/2008-03在解放军第三军医大学烧伤研究所实验室完成.对象:标本取材于解放军第三军医大学西南医院关节外科中心同期收治的10例人工膝关节置换患者软骨组织(＞3 cm).方法:切取原发性骨关节炎患者关节软骨,将软骨剪成1mm×1 mm×1mm左右的碎块,以Ⅱ型胶原酶顺序消化联合胰蛋白酶消化分离关节软骨细胞,同期对比用Ⅱ型胶原酶加胰蛋白酶消化法消化软骨后的收获细胞数及细胞成活率,分离细胞进行原代和传代培养.主要观察指标:以倒置相差显微镜观察体外培养软骨细胞形态;以甲苯胺蓝染色以及Ⅱ型胶原免疫组织化学染色进行软骨细胞鉴定;四甲基偶氮唑盐法测定骨关节炎细胞增殖情况.结果:改良的Ⅱ型胶原酶顺序消化联合胰蛋白酶法,对原代关节软骨细胞的分离取得了优良而稳定的效果,与传统的胰蛋白酶消化法相比,收获细胞数为3.72×106,细胞存活率为97.5%,二者相比有显著性差异(P＜0.05).体外培养观察软骨细胞贴壁和生长均较缓慢.原代和传代后细胞Ⅱ型胶原免疫组织化学和甲苯胺蓝异染反应均较弱.软骨细胞增殖和生长缓慢.结论:Ⅱ型胶原酶顺序消化联合胰蛋白酶分离骨关节炎患者软骨细胞的技术简单且易操作,分离培养的软骨细胞符合骨关节炎时软骨退变的表现,可为骨关节炎的病因研究及软骨细胞移植治疗骨性关节炎提供实验基础.     

预损伤与改良传代体外培养许旺细胞的比较(英文)

背景:通过组织工程方法构建神经桥接体修复神经损伤,需要大量纯化体外培养的许旺细胞。目的:对比观察预损伤法和改良传代法获取许旺细胞的纯度与质量。方法:①预损伤法:预损伤SD乳鼠坐骨神经,3d后取出坐骨神经,分离神经外膜,用胰酶、胶原酶消化,差速贴壁除去成纤维细胞,接种培养。②改良传代法:直接获取SD乳鼠坐骨神经,分离神经外膜,运用双酶消化法结合单酶消化法进行许旺细胞原代培养,5～7d后采用单酶快速消化离心法行传代培养,同时纯化许旺细胞。结果与结论:预损伤法和改良传代法体外培养的许旺细胞纯度均达95%以上,两种方法获得的许旺细胞纯度差异无显著性意义(P>0.05)。两种方法获取的许旺细胞形态正常,数量及纯度高,增殖旺盛,说明预损伤法和改良传代法都是体外获取高质量与高纯度许旺细胞的理想方法。     

两种消化酶在培养晶状体上皮细胞中的消化作用

目的：分析Ⅰ型胶原酶和胰酶在晶状体上皮细胞体外培养中的不同消化作用,以寻找更好的实验方法。方法采用胰酶消化培养法,Ⅰ型胶原酶消化培养法,分别进行晶状体上皮细胞的原代培养,设立对照组和实验组,倒置相差显微镜观察细胞的生长情况,并通过台盼蓝染色观察经消化酶解后游离细胞的存活数量,判别两组间消化作用的差异。结果Ⅰ型胶原酶消化的组织片更容易贴壁,生长速度更快,其游离细胞台盼蓝不着色率为（78．85±6．78）％,胰酶消化法为（27．20±3．28）％,两种酶消化后细胞存活率比较,差异有统计学意义（P＜0．05）。结论Ⅰ型胶原酶消化法培养晶状体上皮细胞的成功率远优于传统改良的胰酶消化法。     

胰蛋白酶及Ⅱ型胶原酶消化获取关节软骨细胞

背景:近年来,众多研究欲采用体外分离培养的关节软骨细胞作为修复缺损的关节软骨的种子细胞,然而,获得纯化的具有生物活性的关节软骨细胞较为困难.目的:拟运用胰蛋白酶与Ⅱ型胶原酶联合消化获取关节软骨细胞.方法:从SD大鼠的正常股骨及胫骨关节表面获取关节软骨,先后运用0.25%的胰蛋白酶和0.2%Ⅱ型胶原酶消化,显微镜下见大量细胞游离后,弃去大块的未消化的关节软骨碎片,离心,去上清,PBS洗涤2次后,加入软骨细胞原代培养液进行培养、增殖.应用甲苯胺蓝及苏木精-伊红染色方法检验所得细胞是否为关节软骨细胞.结果与结论:在严格掌握酶的浓度及消化时间的前提下,通过0.25%胰蛋白酶和0.2%Ⅱ型胶原酶联合酶解关节软骨的方法,成功从大鼠股骨及胫骨关节软骨内分离培养出细胞,并经过甲苯胺蓝染色及苏木精-伊红染色证实,所得的细胞为具有生物活性的关节软骨细胞.     

改良型混合酶消化法培养小鼠乳腺上皮细胞

背景:目前采用的A型胶原酶和胰蛋白酶混合酶分离乳腺细胞团的方法操作复杂,实验条件要求高.目的:观察改良型混合酶消化法能否成功进行乳腺上皮细胞的体外培养.方法:采用Ⅰ型、Ⅱ型胶原酶和胰蛋白酶(1∶1∶1)混合消化直接用眼科剪剪碎的小鼠乳腺组织,37 ℃振荡消化获取乳腺细胞团,并采用差速贴壁法去除成纤维细胞,将细胞团接种于细胞培养瓶进行培养.结果与结论:倒置显微镜下观察显示,改良型混合酶消化法能获得较多的细胞团,且培养12 h后细胞团绝大多数贴在细胞瓶壁,培养72 h后细胞团完全铺开融合成片,细胞呈典型的铺路石样.免疫荧光细胞化学染色结果显示,培养细胞角蛋白18呈阳性反应.说明应用改良型混合酶消化法能在短时间内获得大量较纯的乳腺上皮细胞.     

犬关节软骨细胞的体外分离与培养

背景：自1967年Manning等提出的通过胰蛋白酶和细菌胶原酶联合消化法分离软骨细胞以来,软骨细胞的体外分离培养研究较多,但目前尚无统一标准。目的：研究犬关节软骨细胞在体外分离以及培养的条件,寻求体外扩增软骨细胞较简便、可行、高效的实验方法。方法：取3周龄幼犬关节软骨组织,应用胰酶、胶原酶制定8种消化方法获取软骨细胞,对比不同方法所获取细胞数量及细胞成活率,分离细胞进行原代及传代培养,观察各代软骨细胞形态。结果与结论：在体外分离犬关节软骨细胞的各种方法中,单纯应用Ⅱ型胶原酶消化法获得的软骨细胞数最多,细胞成活率最高。软骨细胞可以通过体外培养获得扩增,并且维持良好的细胞形态及表型,但仅限于5代以内。     

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。     

预损伤与改良传代体外培养许旺细胞的比较

背景：通过组织工程方法构建神经桥接体修复神经损伤,需要大量纯化体外培养的许旺细胞。目的：对比观察预损伤法和改良传代法获取许旺细胞的纯度与质量。方法：①预损伤法：预损伤SD乳鼠坐骨神经,3d后取出坐骨神经,分离神经外膜,用胰酶、胶原酶消化,差速贴壁除去成纤维细胞,接种培养。②改良传代法：直接获取SD乳鼠坐骨神经,分离神经外膜,运用双酶消化法结合单酶消化法进行许旺细胞原代培养,5～7d后采用单酶快速消化离心法行传代培养,同时纯化许旺细胞。结果与结论：预损伤法和改良传代法体外培养的许旺细胞纯度均达95%以上,两种方法获得的许旺细胞纯度差异无显著性意义（P〉0.05）。两种方法获取的许旺细胞形态正常,数量及纯度高,增殖旺盛,说明预损伤法和改良传代法都是体外获取高质量与高纯度许旺细胞的理想方法。     

不同消化酶对体外培养的原代乳鼠雪旺细胞的影响

目的　比较不同消化酶对体外培养的原代乳鼠雪旺细胞的影响。方法　取1周龄SD乳鼠坐骨神经,分别用胰蛋白酶、胶原酶Ⅱ型、混合酶消化,相同方法及实验条件下进行雪旺细胞培养,观察细胞形态、计算2 4h贴壁活细胞百分比及MTT微量比色法比较细胞的生长增殖状况。结果　胰蛋白酶消化组细胞收获量相对最小,细胞损伤程度较大;胶原酶组细胞有一定程度损伤;混合酶组细胞收获量最大,细胞生长状态较好。结论　单纯使用胰蛋白酶或胶原酶消化对原代雪旺细胞的活性及增殖有一定影响,采用胰蛋白酶与胶原酶联合消化效果较好。     

Reliability of four methods for the diagnosis of acute infection by Epstein-Barr virus

We studied the reliability of new indirect tests in the diagnosis of acute infection by Epstein-Barr virus (EBV). Studied for all samples were: method 1, the heterophil antibodies (Abs) (Monolatex, Biokit, Germany); method 2, the IgM Abs to EBV with ELISA tests (antigen pools, Enzygnost, Behringwerke, Germany); method 3, EA (Biotest Diagnostics, Germany); and method 4, the IgG avidity test. The reliability of the four tests for the detection of primary infection by EBV was: sensitivity (method 1:89.1%; method 2: 100%; method 3: 79.7%; method 4: 99%); specificity (method 1: 98%; method 2: 100%; method 3:84%; method 4: 100%); positive predictive value (method 1: 97.6%; method 2: 100%; method 3: 73.6%; method 4: 100% method 4: 100%), and negative predictive value (method 1: 90.7%; method 2: 100%; method 3:84%; method 4: 99%). The IgG avidity test (method 4) is simple and automated in the laboratory and is very useful for ascertaining, from a single sample, the time since infection. It is criteria of recent primoinfection higher levels than 55% of IgG with low avidity for the antigen. The investigation of the Abs to antigen pools (method 2) by ELISA of virus had a high reliability, but the investigation of heterophil Abs by latex (method 1) and the Abs IgM to EA (method 2) were lacking in sensibility regarding their use in the diagnosis of the primoinfection. J. Clin. Lab. Anal. 11:78–81. © 1997 Wiley-Liss, Inc.     

烧伤后膝关节瘢痕屈曲畸形整复16例观察

目的：探讨腘窝烧伤后膝关节瘢痕屈曲畸形的治疗方法。方法：采用手术、理疗和功能锻炼等治疗手段。结果：膝关节功能恢复良好。结论：手术是基本的治疗方法,康复治疗是同样重要的治疗措施。     

气管切开内套管消毒方法的改进

目的：总结气管切开内套管的消毒效果。方法：随机将需行气管切开的总分为两组，甲组内套管用煮沸法，乙组用浸泡消毒法。比较两种方法消毒的效果、程序与优缺点。结果：浸泡消毒法省时、省电、去腥臭，达到了灭菌的效果。结论：浸泡消毒法优于传统的煮沸法，能有效地祛除痰的腥臭味，省时、省电，效果可靠，预防了交叉感染。     

血流切变率的多普勒估计方法

目的：研究血液动力学的一个重要参数—血流剪切率。方法：本文提出了一种利用超声多普勒技术估计血流剪切率的方法，这种方法先估计多普勒信号的平均频率曲线和最大频率曲线，然后计算血流的速度剖面，最后得到时变的血流剪切率。结果：文中还给出这方面研究的实验及结果。结论：本文提出的方法是无损估计血流剪切率的有效方法。     

肠球菌对万古霉素药敏实验方法的探讨

目的 :用 4种药敏试验方法比较肠球菌对万古霉素药敏结果的可靠性。方法 :采用Vitek 32型GPB TG药敏卡、肉汤稀释法、K B纸片扩散法和琼脂筛选法进行球肠菌对万古霉素的药敏实验。结果 :Vitek药敏卡和肉汤稀释法的药敏结果无显著差异 (P >0 .0 5 ) ,K B法药敏结果与上述两种方法比较 ,有非常显著的差异 (P <0 .0 1)。结论 :肠球菌对万古霉素的药敏检测方法最好采用仪器法或肉汤稀释法 ,K B纸片扩散法度及耐药结果不可靠 ,琼脂筛选法检测对万古霉素低水平耐药的肠球菌最灵敏     

Comparing 2 methods of emergent zipper release

BACKGROUND: There are several types of emergent zipper release methods described. The standard method can be difficult. The purpose of this study is to determine if an alternate method of zipper release can be easier to accomplish. METHODS: Subjects were provided with zippers and were taught 2 methods of emergent zipper release using a standard method (cutting the median bar of the actuator) and an alternate method (cutting the closed teeth of the zipper). The elapsed times to successful zipper release for both methods were measured. RESULTS: Mean zipper release times were faster for the alternate method (10.5 seconds) compared with the standard method (75.8 seconds) ( P < .001). CONCLUSION: The alternate method of zipper release is faster and easier than the standard method of zipper release; however, the optimal procedure is also dependent on the location of the entrapped tissue relative to the zipper actuator and the type of zipper.     

Accuracy of FVIII:C assay by one-stage method can be improved using hemophilic plasma as diluent

BACKGROUND: the basic prerequisite of Factor VIII clotting assay (FVIII:C) by One-Stage Method is that all other than FVIII clotting factors are present in constant concentration in each dilution of both standard reference and patient's plasma curves. On the contrary, the plasma content of each dilution is decreasing as the dilution factor increases. OBJECTIVES AND METHODS: to keep exactly constant the plasma content in each mixture, we performed all dilutions of both standard reference and patient's plasma with FVIII deficient plasma and further with a fixed amount of buffer (method B). To show the discrepancies between this method and regular method A, using buffer to make dilutions, a comparative study was conducted on FVIII: C assay on samples at known FVIII concentration and in patients' plasma. Imidazole or Owren's buffers and five different aPTT reagents were employed, both in method A and B. RESULTS: a discrepancy between FVIII: C assays obtained by method A and B was observed, mainly when Pathrontin SL and Imidazole buffer were used. The assays derived from method B always better fit with the expected, calculated, values of FVIII:C concentrations. Furthermore, FVIII: C was assayed in 60 patients: the outcome of method A was always higher than values of method B. The discrepancy between the two methods was higher at FVIII concentrations below 50 U/dL but null at 100 U/dL. The A slope was steeper than B slope and the difference was statistically significant starting from the 1/10 dilution. Accordingly, FVIII: C of patients' plasma obtained by method A was always higher that those obtained by method B, even 2 or 3 times for FVIII level < or = 10 U/dL or 1.4-1.6 times for FVIII levels between 10 and 25 U/dL. CONCLUSIONS: only method B is able to give FVIII: C assays in agreement with the expected values. The dilution of reference standards and samples with FVIII deficient plasma is crucial to accurately evaluate the post-infusion FVIII concentrations in pharmacokinetic studies or the trough level during prophylactic therapy and to investigate the discrepancy among different FVIII: C assays. In addition, the assessment of severity and classification of hemophilia should be reviewed.     

老年病人手部静脉穿刺后固定方法的效果观察

[目的]探讨老年病人手部静脉穿刺后新的固定方法的效果。[方法]对住院接受静脉输液203例老年病人采用传统固定法和新的固定方法,进行自身对照。[结果]新固定方法的漏针率明显低于传统固定方法,病人的满意率明显高于传统固定法,比较有统计学意义(P<0.05)。[结论]采用新固定方法值得在临床应用。     

自动经皮腰椎间盘摘除术的负压研究之二髓核溢出、间盘膨出还纳与负压

目的:探讨髓核溢出、间盘膨出还纳与负压抽吸的不同方法和体位。资料与方法:截取猪腰椎间盘试件60个,用2吨千斤顶加载制成髓核溢出及间盘膨出模型,采取不同方法和体位负压抽吸。结果:髓核溢出:制成模型46个,体位不变时采取五种方法抽吸:切吸常规、注射器抽吸不能使髓核还纳,直接法、切吸常规+关水路、切吸常规+直接法能使髓核不同程度还纳,但还纳率较低。切吸常规+直接法改变6个体位:后伸体位不能使髓核还纳,左曲、右曲、左旋、右旋四个体位能使髓核不同程度还纳,还纳率也较低,前屈体位,能使髓核还纳率达100％,还纳程度也明显优于其它体位·(P(0·01)。间盘膨出:因模型制作困难,所以结果不可靠。结论:切吸常规+直接法前屈体位是提高自动经皮腰椎间盘摘除术髓核还纳率的最佳方向和体位。     

An example of a bootstrap technique application for judging a variability of method performance estimates and its consequences for quality control planning

BACKGROUND: Flexible fitting of quality control procedures to method performance is an inseparable element of a cost-effective quality management strategy. However, opinions on method performance, based on a restricted number of quality control results used to calculate bias and imprecision, can vary due to the random nature of the data-gathering process. METHODS: We present an example of the bootstrap technique application in evaluating method performance estimates. RESULTS: Our analysis reveals substantial variability in method performance assessment results that should not be omitted. We suggest that the best way of using information concerning method performance is to consider the information in a probabilistic manner. Method performance is a feature presented with an element of probability. The effectiveness of a quality control procedure should be assessed with regard to this probability. CONCLUSIONS: Presented example of a bootstrap technique application increases our awareness of the uncertainty that accompanies method performance judgment and quality control planning.