immunophenotyping

求翻译一篇英文化学文献

意思是世界品牌实验室（World Brand Lab）；左边卫（ Wing Back Left）；晶圆背面迭片结构（Wafer Backside Lamination）；网络学习（（Web-based Learning）；宽带激光器（WideBand Laser）。

短语

Alarm indicator WBL 告警灯。

WBL-MBRT 沃尔玛

WBL WideBand Laser 宽带激光器。

Conclusion: Peripheral blood cell preparation of patients with CA and RGH influences the levels of the lymphocyte immunophenotyping. The WBL method is better than the F-PBL method.。

结论：CA和RGH患者外周血不同制备方法影响淋巴细胞免疫表型结果分析，裂解全血法优于分离淋巴细胞法。

紧急，请英语高手帮忙翻译一下。谢谢

求翻译一篇英文化学文献

Quantifying the Cluster of Differentiation 4 Receptor Density on Human T Lymphocytes Using Multiple Reaction Monitoring Mass Spectrometry 。

多反应监测质量光谱法应用于人类T淋巴细胞量化分化抗原簇4受体密度 。

ABSTRACT: Cluster of differentiation 4 (CD4) is an important glycoprotein containing four extracellular domains, a transmembrane portion and a short intracellular tail. It locates on the surface of various types of immune cells and performs a critical role in multiple cellular functions such as signal amplification and activation of T cells. It is well-known as a clinical cell surface protein marker for study of HIV progression and for defining the T helper cell population in immunological applications. Moreover, CD4 protein has been used as a biological calibrator for quantification of other surface and intracellular proteins. However, flow cytometry, the conventional method of quantification of the CD4 density on the T cell surface depends on antibodies and has suffered from variables such as antibody clones, the fluorophore and conjugation chemistries, the fixation conditions, and the flow cytometric quantification methods used. In this study, we report the development of a highly reproducible nano liquid chromatography−multiple reaction monitoring mass spectrometry-based quantitative method to quantify the CD4 receptor density in units of copy number per cell on human CD4+ T cells. The method utilizes stable isotope-labeled full-length standard CD4 as an internal standard to measure 。

endogenous CD4 directly, without the use of antibodies. The development of the mass spectrometry-based approach of CD4 protein quantification is important as a complementary strategy to validate the analysis from the cytometry-based conventional method. It also provides new support for quantitative understanding and advanced characterization of CD4 on CD4+ T cells. 。

摘要：集群分化4(CD4)是一种重要的糖蛋白，它包含四个胞外区域,横跨膜的部分和短的细胞内尾巴。它位于各种类型免疫细胞的表面，在多种细胞功能中扮演重要角色，像细胞信号放大和激活的T细胞。众所周知的是作为研究艾滋病病程的临床细胞表面蛋白标记和在免疫学应用程序中定义辅助T细胞数量。除此之外，CD4细胞蛋白质也已被用作其他表面和胞内蛋白量化的生物校准器。但是，流式细胞，传统量化CD4 T细胞表面密度的方法取决于抗体和并且会受到像抗体克隆、荧光团和结合化学、固定条件以及以前流式细胞定量方法等变量带来的改变。在这项研究中，我们报道一种人类CD4 + T细胞中量化CD4受体密度在每个细胞的拷贝数的高度可再生的纳米液相色谱 - 多反应监测质谱为基础的定量方法的发展。该方法利用稳定同位素标记的全长标准CD4作为内部标准来直接衡量内源性CD4，而不需要使用抗体。 以CD4质谱为基础的蛋白定量方法的发展，作为一个重要的补充战略来验证分析以流式细胞仪为基础的传统方法。它还提供。

了定量的理解和CD4在CD4 + T细胞上高级鉴定的新支持。

Cluster of differentiation 4 (CD4) is a glycoprotein that locates on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells. As a coreceptor, CD4 amplifies the signal generated by the T cell receptor, which is essential for activation of many molecules involved in the signaling cascade of an activated T cell. In human T lymphocytes, CD4 receptor protein is encoded by the CD4 gene1and has four distinct extracellular domains (D1 to D4), a transmembrane portion, and a short intracellular tail.2The use of antihuman CD4 monoclonal antibodies generated against the four extracellular domains has been widely used to define T helper cells in immunophenotyping. Although the number of CD4+ T cells decreases in the progression of HIV-1 viral infection deriving from the gp120 viral protein binding to the CD4 receptor, Poncelet et al. reported that the surface CD4 density still remained constant on T helper cells of HIV-1 infected individuals.3Since then, multiyear research has supported the theory that CD4 expression/density can be used as a biological calibrator for quantification of other surface and intracellular proteins.4 。

分化4 （ CD4）的集群是一种糖蛋白，位于免疫细胞如T辅助细胞，单核细胞，巨噬细胞和树突状细胞的表面。作为一个辅助受体，CD4放大由T细胞受体产生的信号，它对很多分子的活化作用很重要包括信号级联激活T细胞。人类T淋巴细胞， CD4的受体蛋白质是由编码CD4 基因并且有四个不同的胞外结构域（D1到D4） ，跨膜部分和短的胞内尾巴。利用抗人CD4单克隆抗体在4各细胞外结构域的繁殖，已被广泛地被用于在免疫表型上定义辅助性T细胞。尽管CD4 + T细胞的数目在HIV -1病毒感染中减少，HIV -1病毒感染来源于病毒的gp120蛋白结合到CD4受体，蓬斯莱等。报告说，表面CD4的密度在艾滋病毒感染者的T辅助细胞上仍保持不变。从此以后，多年的研究支持了CD4表达/密度可用作生物校准器用于其它表面和细胞内蛋白质量化的这一理论。

Quantitative multicolor flow cytometry incorporating anti- bodies and a fluorescence detection method has played a critical role in clinical diagnostics and immunotherapies. Though the ultimate objective of quantitative flow cytometry is to measure the number of antigens or ligand binding sites associated with a cell, the task is carried out by measuring the number of antibodies bound per cell (ABC). It is critically important to produce biological cell reference materials that bear well-characterized protein markers such as CD4 for the trans-formation of a calibrated linear fluorescence intensity scale of a flow cytometer channel to a biologically meaningful ABC scale.7The quality of the cytometric measurements is affected by variables such as antibody clones, the fluorophore and conjugation chemistries, the fixation conditions, and the flow cytometric quantification methods used.4,8−11Hence, in addition to characterizing candidate reference cell preparations that use antibody-based cytometric methods,12it is necessary to develop a complementary approach to validate the absolute quantification of reference marker proteins such as CD4 without the use of antibodies. 。

定量多色流式细胞结合体和荧光检测方法在临床诊断和免疫治疗中起到了至关重要的作用。虽然定量流式细胞仪最终目标是测量抗原或与细胞结合配体结合位点的数目，该任务的完成是通过测量每个细胞（ ABC）抗体结合的数目。这对于。

如何用流式检测小鼠外周血中T细胞的凋亡

Biphenotypic acute leukemia (BAL) is a rare type of leukemia, and its unique clinical and biological characteristics and prognosis. BAL in the diagnosis and treatment are different from a single type of leukemia. Therefore, we retrospectively analyzed six cases of BAL patients, these patients are in accordance with the European Cooperative Group International Leukemia (EGIL) 1995 standard and the diagnosis of biphenotypic acute leukemia. In this study, we have six cases of BAL specimens of bone marrow cells by light microscopy, respectively, morphological and cytochemical staining to determine their FAB type; the application of flow cytometry immunophenotyping to do, namely, the use of a series of single - Detection of cell membrane antibodies and antigen expression in the cytoplasm; At the same time, the use of G-banding karyotype analysis techniques; the use of both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) treatment in the treatment of four cases in this group of patients with BAL. At present, we have found about the same period of BAL in patients with the diagnosis of acute leukemia of 9.6%. BAL in patients with clinical manifestations in patients with AML and ALL was no significant difference; morphology on the performance of BAL for the AML-M2, M5 and ALL-L3; Immunophenotyping showed that BAL patients with myeloid, B Department of expression were found . In addition, HLA-DR, CD34 positive in the high expression in BAL, suggesting that BAL may be the origin of the leukemic cells in the early hematopoietic cells; karyotype analysis of chromosome abnormalities can be seen, but the study subjects can not be confirmed whether the abnormal karyotype with specificity. BAL in patients with poor response to treatment, the disease risks of poor prognosis. Four cases of treatment of patients with BAL for the first time of chemotherapy complete remission one cases, accounting for 25%. Another in the second two cases of complete remission after chemotherapy, the remaining case of 3,4 for the first course of treatment has yet to ease.。

中性粒细胞(PMN)是机体防御系统的主要组成部分，能吞噬和杀灭多种致病原。致病原与血清中的特异性抗体(IgG)结合后,通过IgG的Fc部分与PMN表面的受体——FcγRⅢ(CD16b)相结合，从而被吞噬。FcγRⅢ有a、b两种类型，FcγRⅢa(CD16a)是一种跨膜糖蛋白，PMN表面的FcγRⅢb(CD16b)是一种糖肌醇磷脂蛋白(GPI-锚蛋白)［1］。近年发现阵发性睡眠性血红蛋白尿症(PNH)患者在血细胞表面缺失各种特异的GPI-锚蛋白，CD16b正是这类蛋白之一。为探讨PMN表面CD16b的缺失与PMN释放氧自由基杀菌功能的关系，将我们的研究报告如下。

材料和方法

1　标本采集　正常人外周血取自协和医院血库正常献血者，正常骨髓取自协和医院胸外科手术患者的肋骨，PNH患者按1987年全国溶血性贫血会议拟定的诊断标准，并经流式细胞仪免疫荧光法检测CD59标记率进一步确诊。

2　中性粒细胞的分离　 新鲜的外周血或骨髓，用肝素抗凝，经淋巴细胞分离液分离，去除淋巴细胞和单个核细胞，用右旋糖酐(dextran，分子量=7～11 万) 沉降和低渗法去除红细胞，得到纯净的中性粒细胞。用台盼蓝染色法检查细胞活力，须保证细胞存活率大于95%。

3　间接免疫荧光法检测CD16　取1×106个细胞，悬浮于100μl 2% BSA中，室温封闭30分钟，加 CD16单克隆抗体(Immunotech SA公司产品)，在室温作用30分钟后，用PBS洗涤2次，再悬浮于100μl 2％BSA中，加FITC 标记的羊抗鼠IgG(Gibco公司产品)室温暗染20分钟后洗去二抗。用500μl PBS悬浮细胞，用流式细胞仪检测CD16 标记率。

4　中性粒细胞功能测定　 佛波醇酯(PMA，Sigma产品，用DMSO溶解，配制成1mg/ml，储存于－20℃，临用前稀释)能激活中性粒细胞，使之释放活性氧，其中O-2*和H2O2能与化学发光剂Luminol(3-氨基苯二甲酰肼，北京化工厂)反应而发光。用LKB-1250发光仪(Luminometer)检测。正常人和PNH患者中性粒细胞，制备成细胞悬液(107 /ml)与 100nmol/L PMA 反应，37℃15分钟,置于冰上终止反应，取含1×105 细胞的激活液加入到1ml 0.1nmol/L 的Luminol 溶液中，立即测定发光值，每一数值测定3次以上， 取平均值。取正常人的不同细胞数，测定发光值，结果显示发光值与细胞数有良好的线性关系，说明此方法具有可靠性 (附图)。

附图　细胞数与发光值的线性关系。

5　细胞培养　以 1×106个细胞/ml的密度接种PMN于IMDM培养基(含10%自身血清)，37℃、5% CO2分别培养12，16，20小时。

6　细胞形态学观察　不同培养时间的细胞用瑞氏染色，光学显微镜下观察。

7　DNA含量分析　取细胞1×106，PBS洗2遍，以70%冷乙醇(4℃)固定12小时以上，用PBS洗去乙醇，加入0.5ml柠檬酸-磷酸缓冲液(pH 7.8)室温作用5分钟，离心后以PBS重悬细胞，加入 RNaseA (终浓度60μg/ml)37℃作用30分钟，再加入碘化乙锭(PI，终浓度50μg/ml)4℃暗染30分钟，以染细胞内总DNA，用流式细胞仪分析正常细胞和凋亡细胞，计算凋亡率。

结果

1　形态学观察　新鲜外周血分离出的中性粒细胞有典型的分叶核，经培养后，形态出现一系列凋亡细胞特性的形态学改变。培养12小时，细胞体积变小，胞浆浓缩，核浓缩；培养16小时以后，部分细胞聚集成团，细胞核碎裂成小块，胞浆中出现空泡，凋亡小体形成。培养时间越长，光镜下可见的凋亡细胞比率越高。

2　中性粒细胞培养过程中CD16的标记率随时间的延长逐渐降低，而凋亡率逐渐增加，中性粒细胞的功能则呈明显的下降趋势，结果见表1。

表1　体外培养不同时间的正常外周血中性粒细胞的几项指标比较。

培养时间

(小时) CD16标率

(%) 流式仪检测

凋亡率(%) 发光频率

(次/分)

0 99.5 1.2 124.32。

12 64.4 29.5 67.27。

16 30.0 39.4 45.43。

20 18.1 64.0 30.18。

3　PNH患者的PMN释放超氧功能与正常人相比有显著的差异，结果见表2。

表2　PNH患者骨髓与正常骨髓中性粒细胞几项指标的比较。

组别 例

数 CD59标记率

(%) CD16标记率

(%) 发光频率

(次/分)

正常组 10 ＞95.00 ＞95.00 117.16±11.47。

PNH组 5 13.60±4.26 2.44±0.98 44.82±16.52。

P值 　 ＜0.001 ＜ 0.001 ＜ 0.001。

讨 论

Ravetch等［1］报告CD16有变异型分布在不同细胞。CD16a分布在巨噬细胞和NK细胞表面，是跨膜蛋白；CD16b仅存于PMN，是GPI锚蛋白。我们应用CD16单抗检测CD16b在PMN表面的量。

PMN的杀菌功能涉及许多方面，有氧化型和非氧化型因素。本实验用不依赖于Fc受体的激活剂PMA来激活PMN，应用发光法测定氧自由基，包括O-2*、H2O2及OH-，测定时，加入PMN激活液即刻连续记录1分钟，以发光最高值计算，代表PMN受刺激后的即刻反应，反映释放氧自由基的能力，氧化型杀菌的功能。

PNH是造血干细胞PIG-A基因突变引起的克隆病，不同患者的异常血细胞GPI-锚蛋白缺失情况有很大差异，Schubert等［2］报道，PNH患者CD16缺失较CD59更明显，我们也观察到这个现象(结果见表2)，而且发现骨髓中PMN CD16b阴性细胞比外周血多，因此选择了PNH患者骨髓的PMN作为研究对象，并且我们所选择的都是病情较严重，病态细胞率高的病例(以CD59的标记率为标准)，因此结果更具有可靠性。

从以上结果得知CD16b表达量低的PNH患者，PMN释放氧的能力亦低，两者有平行的关系。为了进一步证实它们的相关性，应用正常人PMN在体外培养，也获得同样的结果。在培养过程中CD16b逐渐减少，释放氧自由基的功能也降低，两者有相伴关系。Hunizinga等［3］也曾发现在PMN激活的同时有CD16b的丢失。

根据Dransfield等［4］的报道，随CD16b表达量的下降，PMN逐渐凋亡。所以在PMN体外培养的同时又观察了其凋亡的情况。从形态及DNA含量变化，证实在CD16b表达量降低，释放氧自由基功能降低的同时，细胞出现凋亡，凋亡程度与其两项指标降低程度同步。

总之，正常人PMN在凋亡过程中CD16b减少的同时，释放氧自由基的功能也降低；PNH患者CD16b减少，释放氧自由基也减少，至于两者之间有何内在关联，有待进一步研究。但至少从另一个侧面说明PNH患者PMN表面CD16b缺失，使受体介导的特异性识别吞噬病原菌的功能下降；释放活性氧减少以致氧化杀菌功能降低，这两方面可能是PNH患者易受感染的原因。

参 考 文 献

1　Ravetch,JV,Perussia B.Alternative membrane forms of FcγRⅢ (CD16)on human natural killer cells and neutrophils.J Exp Med,1989,170:481-497.。

2　Schubert J,Alvarado M,Ucicechowski P,et al.Diagnosis of PNH using immunophenotyping of peripheral blood cells.Br J Haematol,1991,79:487-492.。

3　Hunizinga TWJ, van der Schoot CE,Jost C,et al.The PI-linked FcγRⅢ is released on stimulation of neutrophils.Nature,1988,333:667-669.。

4　Dransfield I,Buckle AM,Savill JS,et al.Neutrophil apoptosis is associated with a reduction in CD16(FcγRⅢ) expression.J Immunol,1994,152:1254-1263。

原文地址：http://www.qianchusai.com/immunophenotyping.html