免疫磁珠法分选骨髓CD34+细胞:纯度影响因素的分析

背景:免疫磁珠分选技术已成为分选CD34+细胞的主要方法之一,在长期实验中发现诸多因素影响其分离纯度,包括磁珠与细胞孵育时干冰与碎冰的使用、磁珠与细胞孵育及分离时摇床的摇摆方向、红细胞裂解液裂解单个核细胞的使用等.目的:在免疫磁珠分选CD34+细胞过程中分别对上述影响因素进行分析改进,以提高分选CD34+细胞的纯度.设计、时间及地点:细胞学体外对照观察,于2008-07/2009-03在太原市中心医院皮肤科实验室完成.材料:骨髓象筛选正常的人骨髓29份,由太原市中心医院血液科提供,取材均经患者同意.CD34+免疫磁珠为Dynal公司产品.方法:采用淋巴细胞分离液密度梯度离心法分离培养人骨髓单个核细胞,用免疫磁珠法分选骨髓CD34+细胞,对其分选过程中的主要环节如冰块与碎冰、摇床的种类以及红细胞裂解液的使用进行方法改进.方法1:按试剂盒提供方法进行磁珠常规分选CD34+细胞.方法2:在方法1基础上,用碎冰替代冰块进行磁珠与单个核细胞孵育.方法3:在方法2基础上,用万向摇床替代水平摇床.方法4:在方法3基础上,向单个核细胞中预先加入红细胞裂解液裂解残留红细胞.主要观察指标:通过流式细胞仪检测不同方法分选出的CD34+细胞纯度.结果:随着不同环节的逐渐改进,CD34+细胞纯度由(32.7±6.6)%升至(84.5±512)%,方法1,2,3,4所分选出的CD34+细胞纯度逐渐升高,组间方差分析差异显著(F=76.209,P<0.01),Bonferroni法两两比较差异亦有非常显著性意义(P<0.01).结论:碎冰、万向摇床及红细胞裂解液的使用能有效提高免疫磁珠法分选人骨髓CD34+细胞的纯度.     

免疫磁珠分离及流式细胞仪分选纯化外周血CD34+/CD90+干细胞

目的　建立人外周血CD34 细胞及其亚群的纯化分离方法。方法　用干细胞采集仪收集了 3例病人的外周血干细胞 ,应用免疫磁珠分离柱快速分离其中的CD34 细胞 ,随后采用分选型EPICSElite流式细胞仪进一步分选出CD34 /CD90 双阳性早期造血干细胞。结果　免疫磁珠分离纯化后CD34 干细胞的纯度可达 83%～ 95 % ,回收率 5 4 %～ 71% ,活细胞率 >95 %。流式细胞仪分选后的CD34 /CD90 细胞纯度可达 90 %以上 ,回收率在 4 0 %～ 5 0 % ,生存率 >95 %。起始标本中CD34 细胞含量越高 ,纯化所得到的干细胞纯度和回收率越高。两种纯化后的干细胞在形态学上有明显的不同。结论　联合应用免疫磁珠分离和流式细胞仪分选 ,可以较高的回收率快速纯化高纯度的CD34 细胞及其亚群     

免疫磁珠法分选骨髓CD34^＋细胞：纯度影响因素的分析

背景：免疫磁珠分选技术已成为分选CD34^＋细胞的主要方法之一,在长期实验中发现诸多因素影响其分离纯度,包括磁珠与细胞孵育时干冰与碎冰的使用、磁珠与细胞孵育及分离时摇床的摇摆方向、红细胞裂解液裂解单个核细胞的使用等。目的：在免疫磁珠分选CD34^＋细胞过程中分别对上述影响因素进行分析改进,以提高分选CD34^＋细胞的纯度。设计、时间及地点：细胞学体外对照观察,于2008-07/2009-03在太原市中心医院皮肤科实验室完成。材料：骨髓象筛选正常的人骨髓29份,由太原市中心医院血液科提供,取材均经患者同意。CD34^＋免疫磁珠为Dynal公司产品。方法：采用淋巴细胞分离液密度梯度离心法分离培养人骨髓单个核细胞,用免疫磁珠法分选骨髓CD34^＋细胞,对其分选过程中的主要环节如冰块与碎冰、摇床的种类以及红细胞裂解液的使用进行方法改进。方法1：按试剂盒提供方法进行磁珠常规分选CD34^＋细胞。方法2：在方法1基础上,用碎冰替代冰块进行磁珠与单个核细胞孵育。方法3：在方法2基础上,用万向摇床替代水平摇床。方法4：在方法3基础上,向单个核细胞中预先加入红细胞裂解液裂解残留红细胞。主要观察指标：通过流式细胞仪检测不同方法分选出的CD34^＋细胞纯度。结果：随着不同环节的逐渐改进,CD34^＋细胞纯度由（32.7±6.6）%升至（84.5±5.2）%,方法1,2,3,4所分选出的CD34^＋细胞纯度逐渐升高,组间方差分析差异显著（F=76.209,P〈0.01）,Bonferroni法两两比较差异亦有非常显著性意义（P〈0.01）。结论：碎冰、万向摇床及红细胞裂解液的使用能有效提高免疫磁珠法分选人骨髓CD34^＋细胞的纯度。     

人胎盘组织造血干/祖细胞的分离富集

为了探索从胎盘组织中分离富集造血干/祖细胞(HSPC)的标化流程,采用机械法加胶原酶消化法制备人胎盘组织单个细胞悬液,用羟乙基淀粉(6%HES)法从中分离出单个核细胞(MNC),再经免疫磁珠分选法分选出CD34-、CD34+CD38-、CD34+CD38+3个细胞亚群,用流式细胞术对各阶段分选细胞进行表型分析并计算分选细胞的富集度和回收率。结果表明:机械法加胶原酶消化法制备的人胎盘组织单个细胞悬液中单个核细胞(MNC)数达(12.30±3.51)×108,与脐血初始样品所含的MNC数(8.86±5.38)×108比较差异无统计学意义,而其CD34+细胞所占百分率[(3.93±2.31)%]则明显高于脐血[(0.44±0.29)%]。胎盘组织单个细胞悬液经6%HES分离后MNC和CD34+细胞的回收率分别为(45.3±11.7)%和(51.1±9.8)%;MNC经免疫磁珠分选后,其CD34+细胞的纯度和回收率分别为(73.4±14.1)%和(52.7±11.7)%。结论:本实验所建立的"机械法加胶原酶消化法-HES分离MNC-MACS分选目标细胞"的分离纯化方法可从胎盘组织获得高丰度、高富集度、高活性的HSPC,为进一步研究胎盘HSPC提供了比较经济、效果较好的分离富集方案。     

CD271和CD133果真能用于脐血间充质干细胞阳性分选吗?

众多研究表明,脐血中存在间充质干细胞(mesenchymal stem cells,MSC),且脐血获得过程简单,对供者没有损伤,因此可以作为MSC的另一个来源,但脐血间充质干细胞含量极少。本研究探讨CD271和CD133免疫磁珠阳性分选是否也能富集脐血中数量极少的MSC。采用免疫磁珠阳性分选方法从脐血单个核细胞(UCB-MNC)中分离得到CD271+、CD271-、CD133+和CD133-4种细胞;将得到的细胞分别进行培养,以未分选的UCB-MNC为对照,观察各组细胞成纤维细胞集落(CFU-F)形成能力;利用流式细胞术、成骨及成脂肪定向诱导分化对MSC进行鉴定。结果表明:CD271和CD133阳性细胞纯度均达85%,但是CD271+细胞中(99.76±0.08)%表达CD45,(6.24±0.03)%表达CD34,而CD133+细胞99%以上都表达CD34和CD45。阳性细胞培养可见单个成纤维样细胞贴壁生长,但不能形成集落并融合,两种阴性细胞中部分标本(约27%)可形成集落,并能融合传代,与对照组(UCB-MNC)培养成功率相似。将两种阴性细胞培养得到的MSC培养至第3代,表型测定基本一致,即CD34-、CD45-、CD14-、CD29+、CD44+、CD73+、CD90+、CD105+,而且具有成骨、成脂肪分化潜能。结论:CD271和CD133不是完全重合的一群细胞,但绝大多数为造血细胞,不能有效的用以扩增MSC。阴性细胞培养成功率与传统贴壁法相似,说明MSC多存在于CD271和CD133阴性细胞中。     

胎儿骨髓间质干细胞与细胞因子对脐血CD34+细胞扩增作用

目的　探讨胎儿骨髓间质干细胞对CD34 细胞体外扩增的造血支持作用。方法　体外分离、纯化胎儿骨髓间质干细胞 ;Mini MACS免疫磁珠分选 3份脐血CD34 细胞 ;建立胎儿骨髓间质干细胞与CD34 细胞共培养体系 :第 1组为单独CD34 细胞培养 ,第 2组为胎儿骨髓间质干细胞 CD34 细胞共培养 ,第 3组为细胞因子 (干细胞因子 ,白细胞介素 3,Flt3配体 ,血小板生成素 ) CD34 细胞共培养 ,第 4组为胎儿骨髓间质干细胞 细胞因子 CD34 细胞共培养。用流式细胞仪检测不同培养时间的CD34 细胞。结果　胎儿骨髓间质干细胞表达CD2 9,CD4 4 ;免疫磁珠分选CD34 细胞的平均纯度为 97.4 % ;胎儿骨髓间质干细胞 CD34 细胞共培养 2 8d ,有核CD34 细胞仍占有核细胞的 6 .4 3% ;CD34 细胞在胎儿骨髓间质干细胞、细胞因子作用下培养 2 8d ,有核细胞总数、CD34 细胞数分别被扩增 1.6 5× 10 5倍、788倍。结论　胎儿骨髓间质干细胞可有效扩增脐血造血干 /祖细胞。     

致敏小鼠CD4~+ CD25~+调节性T细胞磁珠分选及体外扩增

本研究探讨致敏小鼠CD4+ CD25+调节性T细胞的分选及体外扩增。流式细胞术检测致敏小鼠及正常小鼠体内CD4+ CD25+ Treg细胞水平,免疫磁珠分选方法从小鼠脾细胞中分选出CD4+T细胞、CD4+ CD25+ Treg细胞和CD4+ CD25-T细胞,负载抗CD3/CD28单克隆抗体MACSiBead联合IL-2共同刺激CD4+ CD25+ Treg细胞进行体外扩增培养,用0.4%台盼蓝染色并计数检测细胞的活性,流式细胞术检测分选后细胞纯度、主要表面标记及Foxp3基因的表达。结果表明:致敏小鼠体内CD4+ CD25+ Treg水平较正常小鼠升高(P<0.05)。分选出CD4+ CD25+ Treg细胞纯度平均达到87%,细胞活性大于97%,高表达Foxp3基因。体外扩增2周后细胞数扩增倍数能够达到42倍,CD4+ CD25+ Treg细胞所占比例为85.32%,Foxp3表达由(76.92±1.72)%稍下降至(75.33±2.11)%(P>0.05)。结论:免疫磁珠分选法能够分选出高纯度的CD4+ CD25+ Treg细胞,该分选方法不影响分选靶细胞的细胞活力;体外成功扩增了CD4+ CD25+ Treg细胞,扩增后的CD4+ CD25+ Treg细胞表面标记及Foxp3基因表达无明显改变。     

人骨髓基质细胞协同外源性细胞因子对脐血CD34+细胞的体外扩增作用

目的探讨人骨髓基质细胞(hBMSC)协同以干细胞因子和FL为主的细胞因子对脐血CD34+细胞的体外扩增作用.方法采用免疫磁珠法分选脐血CD34+细胞,以SCF+IL-3+IL-6+FL+EPO组合高效扩增CD34+细胞[1],并结合该细胞因子组合接种到预先照射(20Gy)的hBMSC上,d10结束培养,收获细胞分别作细胞计数、集落培养和流式细胞术检测CD34+细胞数.结果本法获得的脐血CD34+细胞纯度较高(92±0.04)%,在hBMSC组培养的d2,造血细胞几乎都粘附到hBMSC上,随着培养时间的延长,CD34+细胞比例不断下降.hBMSC组与无hBMSC组相比,除细胞总数扩增倍数外,CFU-GM、BFU-E、CD34+细胞扩增倍数差异有显著性意义(P<0.05).结论①脐血来源的CD34+细胞粘附于滋养层上形成造血灶,且10d后造血细胞仍具有体外集落形成能力,表明骨髓基质细胞可支持并维系体外造血;②hBMSC协同外源性细胞因子可能是扩增造血干/祖细胞的较理想方案.     

自体纯化CD34~+细胞移植治疗中/高危淋巴细胞来源恶性肿瘤的临床研究

目的分析自体纯化CD34+细胞移植治疗中/高危淋巴细胞来源恶性肿瘤的临床疗效。方法 10名中/高危组淋巴细胞来源恶性肿瘤患者行自体纯化CD34+细胞移植治疗,细胞分选采用cliniMACS系统。计算并统计分选纯度和CD34+细胞回收率,观察移植相关并发症及患者生存情况。结果纯化后CD34+细胞纯度为(87.79±3.73)%,回收率达到(65.74±10.37)%。10名患者全部顺利造血重建,感染发生率50%(5/10例),复发率为20%(2/10例)。结论利用CliniMACS系统进行外周血CD34+细胞分选,CD34+细胞纯度、回收率均满意,自体移植后造血功能重建顺利,近期疗效满意。     

人脂肪组织来源CD34＋／CD31-细胞向内皮细胞诱导分化的实验研究

目的 探讨将人脂肪组织来源CD34 /CD31-细胞诱导为内皮细胞的可行性.方法 脂肪组织来自5例接受脂肪抽吸术的患者,酶消化法获取原代细胞,细胞扩增培养至第二代,应用免疫磁珠仪分选CD34 /CD31-细胞.将此群细胞用VEGF及bFGF进行诱导.流式细胞仪检测分选前后细胞纯度;免疫细胞荧光检测细胞vWF和CD31的表达;荧光显微镜观察细胞摄取低密度脂蛋白的功能;三维培养观察细胞形成血管样结构的能力.结果分选前后CD34 /CD31-细胞比例分别为(12.57±1.83)%和(96.05±6.73)%;细胞诱导10d后呈现内皮细胞典型的"铺路石"样形态,vWF、CD31表达阳性,细胞具有摄取低密度脂蛋白的功能,三维培养条件下形成"树枝样"分叉结构.结论人脂肪组织来源的CD34 /CD31-细胞能够诱导分化为成熟内皮细胞.     

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.     

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.     

Molecular characterization of virulence and antimicrobial resistance profile of Shigella species isolated from children with moderate to severe diarrhea in northeastern Brazil

Molecular characterization of virulence and antimicrobial resistance profiles were determined for Shigella species isolated from children with diarrhea in Fortaleza, Brazil. Fecal specimens were collected along with socioeconomic and clinical data from children with moderate to severe diarrhea requiring emergency care. Shigella spp. were isolated by standard microbiological techniques, and we developed 4 multiplex polymerase chain reaction assays to detect 16 virulence-related genes (VRGs). Antimicrobial susceptibility tests were performed using disk diffusion assays. S. flexneri and S. sonnei were the predominant serogroups. S. flexneri was associated with low monthly incomes; more severe disease; higher number of VRGs; and presence of pic, set, and sepA genes. The SepA gene was associated with more intense abdominal pain. S. flexneri was correlated with resistance to ampicillin and chloramphenicol, whereas S. sonnei was associated with resistance to azithromycin. Strains harboring higher numbers of VRGs were associated with resistance to more antimicrobials. We highlight the correlation between presence of S. flexneri and sepA, and increased virulence and suggest a link to socioeconomic change in northeastern Brazil. Additionally, antimicrobial resistance was associated with serogroup specificity in Shigella spp. and increased bacterial VRGs.     

护理干预对面部中重度寻常型痤疮的临床疗效影响分析

目的研究护理干预对面部中重度寻常型痤疮的临床疗效影响。方法选取本院在2014年4月~2016年7月诊治的136例面部中重度寻常型痤疮患者,随机分为研究组与对照组,每组68例;所有患者均依据其情况给予对应的治疗,其中对照组在治疗期间给予常规护理,研究组在对照组的基础上再给予综合性护理干预,比较两组的治疗效果及护理满意度情况等。结果患者在接受治疗和护理后,研究组中度与重度患者的治疗效果较对照组均明显提高(P0.05),研究组护理满意度较对照组明显增高(P0.05)。结论对面部中重度寻常型痤疮患者在其治疗期间给予综合性护理干预,具有良好的效果。