冻干血小板聚集和活化的实验研究

目的了解血小板在冻干前、后功能活性的变化。方法应用流式细胞仪检测冻干复水(再水化)血小板及未冷冻干燥血小板胞膜CD61、CD62p、PAC-1的分子表达,评价冻干复水后血小板活化状态;应用血小板聚集仪检测通过诱导剂瑞斯托霉素、二磷酸腺苷诱导的血小板最大聚集率,分析冻干血小板复水后血小板聚集率变化。结果血小板冻干保护剂渗透压值为(2 055.6±123.8)m OSmol/kg;冻干前及复水后血小板分布宽度(PDW)(%)分别为15.50±3.05、18.29±0.55,MPV(f L)分别为9.01±1.84、8.63±0.58;冻干血小板复水回收率(%)为81.57±12.57;冷冻前及复水后血小板最大聚集率(%)分别为34.30±33.14、22.10±23.25;冷冻前及冻干复水后血小板胞膜CD61分子表达(%)分别为43.17±13.55、48.64±13.24,CD62p分子表达(%)分别为17.34±6.47、9.79±4.48,PAC-1分子表达(%)分别为4.92±6.32、4.22±4.64。结论冷冻前及复水后血小板最大聚集率的变化甚微,冻干复水后血小板仍具仍具有一定的存活能力;冷冻前及冻干复水后血小板胞膜CD61、CD62p和PAC-1分子表达略有变化(P0.05)。     

恶性肿瘤患者血小板、红细胞黏附分子CD35、CD44、CD62P的表达与肿瘤转移的关系

摘 要 研究血小板以及红细胞CD35、CD44和CD62P分子的表达变化与肿瘤转移的生物学意义。通过流式细胞仪测定正常人和恶性肿瘤患者血小板和红细胞CD35、CD44和CD62P的表达情况。结果显示，恶性肿瘤患者血小板CD35和CD62P平均荧光强度明显高于正常人，转移组明显高于未转移组（P＜0.05）。转移组血小板CD44平均荧光强度明显高于正常人（P＜0.05）。恶性肿瘤患者红细胞CD35（P＜0.05）和CD44（P＜0.01）平均荧光强度均明显低于正常人，转移组红细胞CD44明显低于未转移组（P＜0.05）。提示恶性肿瘤患者血小板CD35、CD44和CD62P分子处于高表达状态，而红细胞CD35、CD44分子表达减少，和红细胞免疫功能下降有关，这些变化可能对肿瘤的生长和转移具有重要意义。     

腹腔镜复杂肝脏切除术围术期 GP73 和红细胞 CD35、CD58、CD44s 分子及肝肾功能的变化

目的观察腹腔镜复杂肝脏切除术围术期患者高尔基体蛋白（GP）73和红细胞CD35、CD58、标准型CD44（CD44s）分子及肝肾功能的变化规律，探讨腹腔镜复杂肝脏切除术的临床应用价值。方法选取2009年1月至2012年6月采用腹腔镜复杂肝脏切除术治疗的20例患者为观察组，同期进行开腹复杂肝脏切除术治疗的20例患者为对照组，对两组患者术前及术后3、7d的GP73、CD35、CD58、CD44s分子及肝肾功能指标进行定量检测和比较。结果术后3d、7d观察组GP73的水平低于对照组（P均〈0．05）；红细胞CD35和CD58、CD44s分子数量均高于对照组（P均〈0．05）；肝肾功能指标波动幅度也均小于对照组（P均〈0．05）。结论与开腹复杂肝脏切除术比较，腹腔镜复杂肝脏切除术对患者的肿瘤标志物GP73水平的降低更明显，对红细胞CD35和58分子、CD44s水平及肝肾功能的不良影响较小，且恢复速度更快。     

2型糖尿病患者红细胞CD35、CD44s和CD58分子含量的变化及临床意义

目的研究2型糖尿病(T2DM)患者红细胞补体受体1型分子(CR1,即CD35)、CD44s和CD58含量变化及临床意义。方法应用完整细胞酶免疫定量分析法对75名健康对照人及66名T2DM患者进行比较。结果T2DM患者红细胞CD35、CD58含量明显低于正常对照人群,2组差异具有非常显著意义(P<0.0001);CD44s下降不明显(P>0.05);身高和体重影响CD35含量变化。结论红细胞CD35、CD44s和CD58含量减低可能导致T2DM患者红细胞免疫功能低下,它们可能共同参与T2DM的发生发展过程。     

红细胞膜表面黏附分子与红细胞寿命的相关性研究

目的 探讨红细胞寿命与其膜表面蛋白黏附分子的关系,以期建立1种检测红细胞贮存时间的方法 .方法 采集10人(份)健康无偿献血者的新鲜红细胞标本10 ml/份,应用Percoll密度梯度离心法将红细胞分成5个年龄(层),流式检测术检测各分层红细胞膜表面黏附分子CD47、CD44、CD147的表达量;应用SPSS统计软件对...     

银屑病与肿瘤患者红细胞CD35定量与淋巴细胞天然免疫活性相关性分析

[目的]探讨银屑病与肿瘤患者红细胞天然免疫分子CD35数量变化与淋巴细胞天然免疫活性变化的相关性状况。[方法]采用流式细胞仪荧光免疫法测定银屑病及肿瘤患者红细胞CD35数量，用淋巴细胞粘附补体调理过的S180癌细胞株的方法测定银屑病及肿瘤患者淋巴细胞的天然免疫活性。[结果]银屑病患者红细胞CD35数量及淋巴细胞粘附活性明显高于正常人，肿瘤患者红细胞CD35数量及淋巴细胞粘附活性明显低于正常人，银屑病患者红细胞CD35数量与淋巴细胞天然免疫活性之间存在正相关性。[结论]银屑病患者红细胞CD35数量与淋巴细胞天然免疫活性明显亢进，而肿瘤患者红细胞CD35数量与淋巴细胞天然免疫功能都低下，两种疾病免疫发病机理明显不同，而且银屑病患者红细胞CD35数量变化与淋巴细胞天然免疫活性变化呈现正相关，红细胞天然免疫活性变化必然影响到淋巴细胞免疫功能。     

红细胞膜免疫分子在细菌L型败血症中的表达

红细胞失去了细胞核,其表面尚存在广泛表达的膜免疫分子,如CD35(CR1),CD44s及CD58等,在机体生理和病理中起着重要作用.它们具有许多生物学功能,可调节两条补体途径,激活、促进吞噬、免疫复合物清除、T、B细胞活化、发育、归巢、肿瘤转移、细胞激活和信号传递均有重要作用.我们报告细菌L型败血症(SBL)的红细胞免疫分子CD35;CD44s及CD58定量表达的初步结果.     

红细胞老化与补体调节蛋白数量变化

目的通过检测不同老化程度红细胞表面CD55、CD35和CD59数量,来探索老化红细胞清除机制中补体调节蛋白所发挥的作用。方法利用流式细胞术,检测30份标本其毛细管分离的年轻、年老红细胞表面CD35数量,和21份样本年轻、年老红细胞表面CD55和CD59数量。另外检测了其中随机选取的3份标本的4℃保存红细胞和37℃保存红细胞表面CD35、CD55和CD59数量。通过流式细胞术检测了15份标本自身IgG抗体的敏感性。结果与毛细管分离的年轻红细胞相比,年老红细胞CD35的平均荧光强度(MFI)下降了约35%(P<10-22)。年轻、年老红细胞表面CD55和CD59数量也呈现显著性下降(CD55:下降约44.17%;CD59:下降约36.18%,P<10-12)。体外保存的红细胞结果有所不同。对于CD35,4℃和37℃保存的红细胞表面数量变化不大无显著性统计学意义(P>0.05)。对于CD55和CD59,37℃保存的红细胞表面数量显著降低(P<0.01),而4℃保存的红细胞数量无显著性差异(P>0.05)。另外,毛细管分离得到的年老红细胞结合的自身抗体IgG显著高于年轻红细胞,荧光强度是年轻红细胞的1.8倍(P<0.01)。结论年轻、年老红细胞自身IgG抗体敏感性不同,说明毛细管离心方法可以一定程度反映人体生理条件下红细胞的老化状态。随着红细胞在血液循环中的老化,3种补体调节蛋白的显著下降,可能会使更多的C3b在红细胞表面沉积,从而中和调理、增强吞噬。此外,体外红细胞的老化与生理条件下的老化可能存在不同机制。     

红细胞CD58在健康施灸人群中的表达:40例变化分析

背景:CD58是一种糖蛋白,属于免疫蛋白超家族成员,广泛表达于人体各种免疫细胞和红细胞,是红细胞调控T细胞免疫功能的重要天然免疫物质基础.目的:通过在神阙、关元等传统保健穴上施灸,观察对健康人群红细胞CD58表达的影响.设计、时间及地点:前后对照、调查分析.于2008-03/12在山西中医学院实验中心完成.对象:选择山西中医学院在校健康大学生40例,男女各20例,平均年龄21.08岁.受试者均知情同意.方法:将以熟地、山药、山茱萸等为主方的药物制成药饼置于受试者神阙、关元、足三里、脾俞、肾俞等穴施灸,隔日1次,共灸10次,进行施灸前后的对比.主要观察指标:在施灸前、后分别进行血细胞的常规检查,流式细胞仪测定红细胞表面CD58分子的阳性百分率和平均荧光强度.结果:施灸后红细胞、白细胞数量较施灸前显著升高(均为P<0.01);与施灸前相比,红细胞CD58分子的阳性百分率和平均荧光强度均明显高于施灸前(均为P<0.01).其中,红细胞CD58荧光强度的变化尤为突出.结论:红细胞CD58分子数量表达与艾灸密切相关,隔药饼灸增强了机体的免疫调节功能.     

冻干前后红细胞血型及生物活性指标的检测

目的检测冻干前、后红细胞血型抗原及其生物学活性,了解冻干前、后红细胞血型抗原及其生物学活性的变化情况。方法红细胞血型抗原的检测采用血型血清学方法,2,3-DPG和ATP用ELISA法。结果在ABO、Rh、MNSs、Kell、Duffy、P血型抗原中,冷冻前、冻干后红细胞血型抗原一致;在Lewis血型抗原中,冷冻前、冻干后红细胞血型抗原有变化。冷冻前、冻干后红细胞2,3-DPG和ATP含量均无明显变化。结论冷冻前、冻干后红细胞Lewis血型系统中的Lea和Leb血型抗原变化有明显差异;冷冻前、冻干后红细胞2,3-DPG和ATP含量变化无统计学意义。     

In vitro RBC exposure to Plasmodium falciparum has no effect on RBC antigen expression

Severe malarial anaemia is a leading cause of death in African children younger than 3 years of age who are infected with Plasmodium falciparum. The pathogenesis of this anaemia is not understood. The purpose of this study was to determine if P. falciparum induces changes in RBC membranes that contribute to the immune destruction of RBCs. RBCs were collected from healthy subjects and tested using standard haemagglutination assays for 45 antigens representing 21 blood group systems/collections before and after exposure to P. falciparum, strain FVO. Lectins were used to determine whether crypt or neoantigens were expressed on the RBC membrane. Polybrene was used to detect changes in sialic acid. RBCs were cultured in vitro with and without the parasite, and blinded serologic studies were completed. CD35 (complement receptor 1), CD55 (decay-accelerating factor), CD59 (membrane inhibitor of reactive lysis) and CD47 (integrin-associated protein) flow cytometric assays were compared for infected and uninfected RBCs. The percentage of parasitaemia was determined using Giemsa-stained thin blood films. Two (Ch, Lub) of the 45 antigens had differing strengths of agglutination between infected and uninfected RBCs, but these differences were resolved with a second source of antisera. Forty-three antigens showed no significant differences in the strength of agglutination between the infected and uninfected RBCs. Lectin and polybrene testing showed no differences. CD35, CD55, CD59 and CD47 levels showed no significant differences. P. falciparum does not appear to alter the expression of classified immunogenic antigens on the RBC membrane in this in vitro system. The pathogenesis of the haemolytic episode that occurs in these children remains unclear.     

Red blood cell age determines the impact of storage and leukocyte burden on cell adhesion molecules, glycophorin A and the release of annexin V.

The influence of the age of the red blood cell (RBC) within its 120-day lifecycle at the time of blood donation on the RBC storage lesion is not well understood. Expression of cell adhesion molecules (CAMs) (CD44, CD47, CD58 and CD147), glycophorin A (GPA) and phosphatidylserine (PS) on young and old RBCs density separated prior to storage of the RBC concentrate was determined by flow cytometry. Older RBCs showed significantly reduced expression of GPA throughout storage and CD44 and CD147 from Day 28 onwards compared to young RBCs. Storage in the presence of leukocytes caused a significant decline in the expression of CD44, CD58, CD147 and GPA, whereas RBCs that were pre-storage leukocyte depleted maintained a relatively consistent level of expression throughout storage. PS was not detected at the external RBC membrane of young or old RBCs during storage. Increased levels of annexin V were detected in the supernatant of RBCs stored in the presence of leukocytes, with significantly greater supernatant levels found for old RBCs compared to young RBCs. These findings provide new insight into the RBC storage lesion and indicate that RBC age at the time of donation impacts upon the quality of stored RBC concentrates.     

Reduced expression of CD47 during murine red blood cell (RBC) senescence and its role in RBC clearance from the circulation

BACKGROUND: Almost 2 percent of murine blood red blood cells (RBCs) are destroyed each day and are replaced by fresh RBCs generated through the process of erythropoiesis. RBCs to be destroyed are phagocytosed by macrophages in the reticuloendothelial system, especially in the spleen. CD47 molecules on RBCs may regulate the susceptibility of RBC to destruction by phagocytosis because its recognition by inhibitory receptor (signal regulatory protein alpha) on macrophages sends a negative signal, which if sufficiently strong, may abort the phagocytic response altogether. The aim of this study was to investigate whether age-dependent changes in CD47 expression on circulating RBCs have a role in destruction of senescent RBCs by macrophages. STUDY DESIGN AND METHODS: A two-step in vivo biotinylation method for labeling mouse RBCs in vivo was used to track the CD47 expression levels as well as the turnover of circulating RBCs of defined age groups. RESULTS: Our results indicate that CD47 expression levels decrease on circulating RBCs throughout their life span in circulation. The oldest RBCs in circulation have 30 percent lower mean expression of CD47 than the youngest RBCs. Depletion of macrophages by administration of clodronate-loaded liposomes resulted in a significant decrease in the mean expression of CD47 on RBCs of all age groups and a significant accumulation of senescent RBCs in blood and spleen. A decrease in mean expression of CD47 and accumulation of senescent RBCs in macrophage-depleted mice were significantly higher for spleen RBCs compared to blood RBCs. CONCLUSIONS: Our results provide supportive evidence for a role of decreasing CD47 expression on aging circulating RBCs in their destruction by macrophages.     

Expression levels of CD47, CD35, CD55, and CD59 on red blood cells and signal-regulatory protein-alpha,beta on monocytes from patients with warm autoimmune hemolytic anemia

BACKGROUND: Animal models have shown that CD47-deficient mice develop severe autoimmune hemolytic anemia (AIHA) because the binding of red blood cell (RBC) CD47 to signal-regulatory protein (SIRP-α) on macrophages contributes to the inhibition of phagocytosis. In contrast, complement-inhibitory proteins such as CD35, CD55, and CD59 may protect RBCs against the lysis by complement.
STUDY DESIGN AND METHODS: With the use of flow cytometric analyses, the expression of CD47, CD35, CD55, and CD59 on RBCs and of SIRP-α,β on peripheral monocytes of 36 patients with warm AIHA (wAIHA; 23 with active wAIHA, 13 with wAIHA in remission) and 20 healthy subjects was evaluated.
RESULTS: The mean fluorescence intensities (MFIs) of the expression of CD47, CD35, CD55, and SIRP-α,β of active wAIHA patients, wAIHA in remission, and healthy subjects were not statistically different. Patients with active wAIHA showed significantly lower CD59 expression on RBCs than healthy individuals (MFI, 512.5 ± 59.6 vs. 553.7 ± 36.6; p = 0.009), while the CD59 expression in patients with wAIHA in remission was not significantly different from that of healthy controls (MFI, 538.4 ± 48.3 vs. 553.7 ± 36.6; p > 0.05). The expression of CD59 on RBCs of 3 patients who died from the wAIHA was lower than that seen on RBCs of healthy controls (MFI, 433.6 ± 69.6 vs. 553.74 ± 36.6; p = 0.0001).
CONCLUSIONS: Our data show that the expression of CD47 on RBCs and SIRP-α,β on monocytes of patients with wAIHA is not different from that seen in healthy individuals. In addition, we detected that patients with active wAIHA present low expression of CD59 and normal expression of CD35 and CD55 on their RBCs. Complement-regulatory proteins may play an important role in protecting RBC destruction through the activation of complement.     

海藻糖负载对人红细胞冷冻干燥保存影响的实验研究

目的初步探讨红细胞冻干长期保存的有效方法,并比较冻干前海藻糖的负载与否对红细胞冻干保存效果的影响。方法实验设实验组(负载海藻糖冻干-复水后红细胞):37℃,红细胞负载海藻糖7h后,采用主要成分为含有15%聚乙烯吡咯烷酮(PVP)和150mmol/L,海藻糖的缓冲液作为保护液,在设定的降温程序下冻干保存红细胞;对照组:未负载海藻糖冻干-复水后红细胞。冻干结束后用37℃的再水化液快速水化,检测2组的各项理化指标。结果实验组红细胞冻干再水化后RBC和Hb回收率要高于对照组(P<0.05);ATP酶和葡萄糖-6-磷酸脱氢镁(G-6-PD)活性水平显著差异有统计学意义(P<0.05))。结论胞内海藻糖对红细胞冻干具有明显的保护作用,红细胞在37℃孵育7h的条件负载海藻糖后冻干-复水后能保持细胞的理化稳定性。     

Expression of CD47 (integrin-associated protein) decreases on red blood cells during storage.

In light of recent studies that demonstrate that CD47 antigen is an important "self-recognition" marker involved in protecting circulating red blood cells (RBCs) from phagocytosis by macrophages, the aim of the present study was to investigate whether CD47 expression on RBCs is altered during storage. Red cell concentrates (RCCs) were prepared and stored at 4 degrees C and samples were collected on days 1, 14, 28 and 42 post donation. RBCs were labelled with anti-CD47 antibody and analysed by flow cytometry. A small but significant and progressive decrease in CD47 antigen expression was observed in non-irradiated and irradiated RBCs stored for 14 days onwards. CD47 was also detected in increasing quantities in supernatant from RCCs after 14 days of storage. It is proposed that loss of CD47 during storage, in addition to other biophysical changes, may target many transfused RBCs for clearance from the transfusion recipient's circulation.     

Immunological impact of the CD71+ RBCs: A potential immune mediator in transfusion

Donor - recipient sex - mismatched transfusion is associated with increased mortality. The mechanisms for this are not clear, but it may relate to transfusion-related immunomodulation. Recently, CD71⁺ erythroid cells (CECs), including reticulocytes (CD71⁺ RBCs) and erythroblasts, have been identified as potent immunoregulatory cells. The proportion of CD71⁺ RBCs in the peripheral blood is sufficient to play a potential immunomodulatory role. Differences in the quantity of CD71⁺ RBCs are dependent on blood donor sex. The total number of CD71⁺ RBCs in red cell concentrates is also affected by blood manufacturing methods, and storage duration. As a component of the total CECs, CD71⁺ RBCs can affect innate and adaptive immune cells. Phagocytosed CECs directly reduce TNF-α production from macrophages. CECs can also suppress the production of TNF-α production from antigen presenting cells. Moreover, CECs can suppress T cell proliferation thorough immune mediation and / or direct cell-to-cell interactions. Different in their biophysical features compared to mature RBCs, blood donor CD71⁺ RBCs may be preferential targets for the macrophages. This report summarizes the currently literature supporting an important role for CD71⁺ RBCs in adverse transfusion reactions including immune mediation and sepsis.     

化疗联合G-CSF动员的骨髓及外周血CD34+细胞表达粘附分子的变化

探讨化疗合G－CSF动员前，后患骺髓及外周血CD34^ 细胞表达CD44，CD49d，CD62L及趋化因子CXCR4的动态变化，用免疫荧光直接三角标记和流式细胞术测定G－CSF动员前，后骨髓及外周血CD34^ 细胞的CD44，CD49d，CD62L及趋化因子CXCR4的表达，观察输注各表达亚群细胞数与移植后造血重建的关系。结果发现，动员后骨髓中CD34^ CD44^ 和CD34^＋CD49d^ 细胞的百分比较动员前明显降低, 而外周血中二的比例则显升高；动员前，后骨髓中CD34^＋CD62L^＋和CD34^＋CXCR4^ 细胞的变化并不明显，而外周血中前明显增加，后则显减少。输入CD44^＋，CD49d^ ，CD62L^ 及CXCR4^ 的CD34^＋细胞的量与移植后血中中性粒细胞和血小板恢复的时间未表明有显的相关，结论：G－CSF动员可下调骨髓CD34^＋细胞的CD44，CD49d，CD62L及CXCR4的表达，从而进入外周血循环，输注这些细胞的临床意义有待累积更多病例的分析。     

A novel mouse model of red blood cell storage and posttransfusion in vivo survival

BACKGROUND: Storage of red blood cells (RBCs) is necessary for an adequate blood supply. However, reports have identified potential negative sequelae of transfusing stored RBCs. An animal model would be useful to investigate the pathophysiology of transfusing stored RBCs. However, it has been reported that storage of rat RBCs in CPDA-1 resulted in an unexpected sudden decline in posttransfusion survival. A mouse model of RBC storage and transfusion was developed to assess survival kinetics of mouse RBCs.
STUDY DESIGN AND METHODS: RBCs expressing green fluorescent protein were collected in CPDA-1, filter leukoreduced, adjusted to a 75% hematocrit, and stored at 4°C. At weekly intervals, stored RBCs were transfused into C57BL/6 recipients. RBC survival was measured by flow cytometry and chromium-51 labeling. Phosphatidylserine externalization and CD47 expression was also evaluated.
RESULTS: Mean 24-hour survivals of transfused RBCs were 99, 91, 64, 54, 30, and 18% after 0, 7, 14, 21, 28, and 35 days of storage, respectively. Stored RBCs showed an initial rapid clearance with subsequent extended survival. Increased surface phosphatidylserine and decreased CD47 expression were also observed.
CONCLUSIONS: Mouse RBCs showed a progressive decline in survival, as a function of storage time, unlike the precipitous loss of viability reported for rat RBCs. Moreover, changes in the measured surface markers were analogous to trends reported for human RBCs. Together, these findings provide an initial characterization of a novel mouse model of RBC storage with the potential to serve as an experimental platform for studying the pathophysiologic consequences of transfusing stored RBCs.