G-CSF动员的骨髓及外周血干细胞移植治疗重型再生障碍性贫血

采用G CSF动员的骨髓以及外周血造血干细胞移植治疗重型再生障碍性贫血 (SAA)。共有 10例重型再生障碍性贫血患者接受了G CSF动员的骨髓以及外周血造血干细胞移植 ,回顾性分析植入情况、植入速度及急慢性移植物抗宿主病 (GVHD)发生率等。所有患者移植后均获得造血重建。中性粒细胞计数 (ANC) >0 5× 10 9L- 1 的中位时间为 13 2d(8～ 18) ,血小板 >2 0× 10 9L- 1 的中位时间为 2 2 2d(10～ 10 8)。 8例获得异体植入的患者中 3例发生Ⅰ Ⅱ度皮肤急性GVHD ,发生 5例慢性GVHD。至随访截止 ,中位随访 775d(2 10～ 14 2 9) ,所有 10例患者均无病存活(DFS)。因此我们认为G CSF动员的骨髓及外周血干细胞移植是治疗SAA的有效方法 ,可获得快速、持久植入 ,而不增加急性和慢性GVHD的发生率。     

外周血干细胞动员调节CD34^＋细胞β整合素及L—选择素的表达

目的：观察造血干细胞表面粘附分子的变化在外周血干细胞动员中的作用。方法：用双色免疫荧光法研究15例恶性血液病患者经化疗＋G－CSF动员外击血干细胞前后、骨髓和外周血CD34^ 细胞表面β1整合素（CD49d）、β2整合素（CD11a、CD11b）及L－选择素（CD62L）的表达。结果：①动员后第7天CD34^ 细胞表面较动员前CD49d、CD11a、CD62L表达下降;（P＜0．01）,而CD11b无变化（P＞0．05）。②外周血CD34^ 细胞表达CD49d、CD11a、CD11b、CD62L较骨髓低（P＜0．05）。③外周血CD34^ 细胞绝大多数处于GO／G1期,该期的的干细胞CD49d的密度低于S＋G2／M期。结论：化疗＋G－CSF通过粘附分子的变化而使造血干细胞从骨髓进入外周血中。     

内皮祖细胞与肿瘤血管新生

血管内皮祖细胞（EPCs）是内皮细胞的前体细胞,特异性表达CD34,CD133和VEGFR-2,具有向血管内皮细胞分化的潜能。EPCs主要位于骨髓和外周血。肿瘤的生长和转移依赖于肿瘤血管新生。肿瘤细胞可合成和释放多种细胞因子,在不同因子的趋化作用下EPCs从骨髓动员至外周血循环,然后迁移和定居到肿瘤组织,经细胞因子诱导分化为成熟内皮细胞,参与肿瘤血管新生。VEGF/VEGFR-2信号途径在EPCs参与的肿瘤血管新生方面起重要作用。     

外周血干细胞动员调节CD34+细胞β整合素及L-选择素的表达

目的观察造血干细胞表面粘附分子的变化在外周血干细胞动员中的作用。方法用双色免疫荧光法研究15例恶性血液病患者经化疗+G-CSF动员外周血干细胞前后、骨髓和外周血CD34+细胞表面β1整合素(CD49d)、β2整合素(CD11a、CD11b)及L-选择素(CD62L)的表达。结果①动员后第7天CD34+细胞表面较动员前CD49d、CD11a、CD62L表达下降(P＜0.01),而CD11b无变化(P＞0.05)。②外周血CD34+细胞表达CD49d、CD11a、CD11b、CD62L较骨髓低(P＜0.05)。③外周血CD34+细胞绝大多数处于GO/G1期,该期的干细胞CD49d的密度低于S+G2/M期。结论化疗+G-CSF通过粘附分子的变化而使造血干细胞从骨髓进入外周血中。     

短程大剂量格拉诺赛特动员自体外周血造血干细胞移植治疗急性白血病

目的 报导短程大剂量格拉诺赛特(rhG—CSF)动员外周血造血于细胞,进行自体外周血造血干细胞移植(APBSCT)治疗急性白血病.方法rhG—CSF,500μg/d皮下注射4天,第5、6天单采外周血单个核细胞(MNC),行CFU—GM培养,CD34~ 细胞计数.予MAC方案预处理.回输自体外周血造血干细胞(APBSC).结果 动员后CFU—GM明显增高,CD 34~ 细胞增多.骨髓造血功能重建迅速.     

内皮前体细胞促骨髓间充质干细胞向肝样细胞转化中的VEGF-Notch信号通路

背景:在前期研究中发现骨髓血管内皮前体细胞在体外培养过程中通过旁分泌的方式产生了能够促进骨髓间充质干细胞分化的因子,血管内皮生长因子是其中之一.目的:探讨骨髓血管内皮前体细胞促进骨髓间充质干细胞向肝样细胞转化中是否有VEGF-Notch信号通路参与.方法:①分离培养SD大鼠骨髓间充质干细胞和骨髓血管内皮前体细胞,将第3...     

rhTPO对巨核系的定向分化效应

目的 :探讨重组人促血小板生成素 (rhTPO)对外周造血干细胞中巨核系祖细胞的定向诱导分化能力。方法 :经化疗及G CSF动员后分离的外周造血干细胞 ,进行液体和集落培养 ,研究rhTPO单独及与IL 3、IL 6、SCF的协同作用。结果 :液体培养后 ,TPO组单个核细胞数 (MNC)扩增了 ( 1.73± 0 .49)倍 ,IL 3 IL 6 SCF组MNC比种植时增加 ( 4.2 0± 1.14)倍 ,IL 3 IL 6 SCF TPO组MNC增加了 ( 4.5 3± 1.2 7)倍。单独应用TPO及TPO与IL 3、IL 6、SCF合用产生高比例的CD41a 细胞 ,TPO组培养前CD41a 细胞为 11.70 %± 5 .2 3% ,培养后CD41a 细胞为 19.17%± 6 .2 6 % ( P <0 .0 5 )。CD41a 细胞数在TPO组扩增了 ( 3.5 2± 1.18)倍 ,IL 3 IL 6 SCF组扩增了 ( 5 .32± 1.79)倍 ,IL 3 IL 6 SCF TPO组扩增了 ( 6 .94± 2 .19)倍。结论 :TPO可以定向诱导巨核系细胞的分化 ,IL 3、IL 6、SCF可以协同TPO的作用 ,这在造血调控研究 ,体外定向扩增外周血干细胞 ,促进外周造血干细胞移植患者血小板的恢复具有应用前景     

26例动员外周血干细胞的程控降温冷冻保存效果评价

目的评价程控降温冷冻保存高浓度外周血干细胞的效果。方法采集动员的患者外周血干细胞,用程控降温系统降温至-80℃后保存于液氮中。冷冻前和解冻后均做有核细胞计数、粒单-集落形成单位(colonyformingunit-granulocyte/macrophage,CFU-GM)培养和苔盼蓝拒染率实验。结果冷冻的外周血有核细胞终浓度平均为1.405×108/ml,融化后有核细胞和CFU-GM回收率分别为80.64%±11.51%和94.01%±25.42%;拒染率为84.69%±8.72%。26例患者除1例死于预处理毒性不能进行评价外,余者均获得快速稳定造血功能重建。结论程控降温冷冻保存高浓度的外周血干细胞是一种安全有效的方法。     

外周血中血管内皮前体细胞的分离和鉴定

新血管的形成主要有两种方式,一是血管形成(vasculogenesis),即造血血管母细胞(hemangioblast)原位分化成内皮细胞并形成原始毛细血管丛的过程;二是血管新生(angiogenesis),指由已存在的血管以出芽的方式形成新血管的过程。一直以来,人们认为血管形成只发生于胚胎发育时期,在出生后并不存在。1997年,Asahara等发现在人的外周血中存在血管内皮前体细胞(endothelial progenitor cells,EPC),     

循环内皮细胞：评价病理状态下血管内皮功能的重要生物标记物

1前言 循环内皮细胞（circulating endothelial cells,CEC）是指能够在外周血中测得的血管内皮细胞（vascular endothelial cells,VEC）。血管内皮细胞具有多种生理功能,参与机体血管的舒缩、凝血、炎症、免疫、物质转运和生物活性物质释放等多种生命活动。正常状态下,内皮细胞每天的更新率大约0％～1％,因此正常人体内CECs的数量非常少,然而在内皮受损的病理状态下,循环内皮细胞会发生数量和形态学的变化。     

阿托伐他汀钙对高血压大鼠血管内皮细胞的保护作用

目的:观察高血压状态下阿托伐他汀钙对SD大鼠胸主动脉血管内皮细胞的保护作用。方法:构建两肾一夹SD大鼠肾性高血压模型。研究动物随机分为高血压组、他汀治疗组和假手术组,每组8只,术后第4周他汀治疗组给予阿托伐他汀钙20mg·kg-1.d-1灌胃8周。术后第4、12周测血压、血脂,第12周通过扫描电镜方法观察胸主动脉内皮细胞层的完整性,检测血液中循环内皮细胞数量,并测定循环内皮祖细胞(CEPCs)的数量及增殖能力、黏附能力及凋亡情况。结果:他汀治疗组大鼠胸主动脉内皮细胞受损轻于高血压组,但重于假手术组,高血压组、他汀治疗组和假手术组大鼠循环内皮细胞数量(5.9×106、3.9×106、2.0×106)与CEPCs凋亡率(22.1%±2.1%、13.4%±1.6%、7.4%±1.3%)依次降低(均P0.01);而CEPCs数量(21.63±2.33、40.38±6.00、65.38±2.97)、CEPCs增殖能力(0.13±0.01、0.17±0.01、0.29±0.03)与CEPCs黏附能力(12.25±2.49、21.50±2.20、28.88±2.85)依次增高(均P0.01)。结论:(1)高血压状态可导致大鼠血管内皮细胞严重受损,CEPCs数量增加,而CEPCs数量与功能降低,凋亡率增高,继而引起CEPCs对血管内皮细胞的修复能力降低。(2)阿托伐他汀钙对血管内皮细胞具有直接保护作用,并可能通过提高CEPCs数量及功能、降低CEPCs凋亡、增强CEPCs对血管内皮细胞的修复能力而发挥作用。     

Changes in circulating endothelial progenitor cells predict responses of multiple myeloma patients to treatment with bortezomib and dexamethasone

Four cycles of chemotherapy are required to assess responses of multiple myeloma (MM) patients. We investigated whether circulating endothelial progenitor cells (cEPCs) could be a biomarker for predicting patient response in the first cycle of chemotherapy with bortezomib and dexamethasone, so patients might avoid ineffective and costly treatments and reduce exposure to unwanted side effects. We measured cEPCs and stromal cell-derived factor-1α (SDF-1α) in 46 MM patients in the first cycle of treatment with bortezomib and dexamethasone, and investigated clinical relevance based on patient response after four 21-day cycles. The mononuclear cell fraction was analyzed for cEPC by FACS analysis, and SDF-1α was analyzed by ELISA. The study population was divided into 3 groups according to the response to chemotherapy: good responders (n=16), common responders (n=12), and non-responders (n=18). There were no significant differences among these groups at baseline day 1 (P>0.05). cEPC levels decreased slightly at day 21 (8.2±3.3 cEPCs/μL) vs day 1 (8.4±2.9 cEPCs/μL) in good responders (P>0.05). In contrast, cEPC levels increased significantly in the other two groups (P<0.05). SDF-1α changes were closely related to changes in cEPCs. These findings indicate that change in cEPCs at day 21 in the first cycle might be considered a noninvasive biomarker for predicting a later response, and extent of change could help decide whether to continue this costly chemotherapy. cEPCs and the SDF-1α/CXCR4 axis are potential therapeutic targets for improved response and outcomes in MM patients.     

Mobilization of circulating endothelial progenitor cells after endovascular therapy for ruptured cerebral aneurysms

Emerging evidence shows that circulating endothelial progenitor cells (EPCs) promote regeneration of the endothelium at sites of vessel injury. This study was designed to test the hypothesis that EPCs are mobilized in patients who had ruptured cerebral aneurysm (CA) and underwent endovascular therapy. Fourteen patients with ruptured CAs were recruited and blood samples were analyzed after coil embolization surgery. Blood samples were also obtained from 18 healthy control subjects who had no evidence of CAs and did not undergo endovascular surgery. We measured the numbers of circulating EPCs, levels of plasma vascular endothelial growth factor (VEGF) and platelet counts. EPCs significantly increased in patients with ruptured CAs and underwent surgical coil embolization, reaching a peak level on day 14 post operation. The levels of plasma VEGF and platelet counts also significantly increased in parallel with the increase in EPCs, leading to significant positive correlations of circulating EPCs with VEGF in plasma (r = 0.636, P < 0.01) and platelet counts (r = 0.721, P < 0.001), respectively. The finding suggests that EPCs are mobilized upon surgery and may play a critical role in repairing injured vascular endothelium. Levels of EPCs in peripheral blood could also serve as a prognostic marker for the outcomes of ruptured cerebral aneurysms after surgical repair.     

糖尿病视网膜病变患者血浆Hcy、CEC和ET水平的检测分析

目的:分析糖尿病视网膜病变患者血浆同型半胱氨酸(Hcy)、外周血循环内皮细胞(CEC)和血浆内皮素(ET)水平及其相互关系。方法:将80例2型糖尿病(T2DM)患者分为2组:无糖尿病视网膜病变(NDR)组50例,糖尿病视网膜病变(DR)组30例,以我院健康体检正常者30例为对照(NC)组。分别测定Hcy、CEC、ET水平。结果:T2DM患者Hcy、CEC和ET水平均明显高于对照组(P<0.01);DR组Hcy、CEC、ET明显高于NDR组(P<0.01)。DR组血浆CEC计数和ET浓度呈正相关(r=0.839,P<0.05),Hcy浓度与CEC计数呈正相关(r=0.615,P<0.05),Hcy与ET浓度亦呈正相关(r=0.642,P<0.05)。结论:T2DM患者血浆Hcy、CEC、ET水平升高与DR有密切关系,血浆Hcy升高及内皮损伤可能在糖尿病患者视网膜病变的发生和发展中起重要作用。     

脑缺血再灌流家兔血液中循环内皮细胞变化及针刺对其的影响

利用缺血再灌流家兔模型研究变化规律，并探讨了脂质过氧化物与循环内皮细胞之间的关系，以期了解缺血再灌流损伤血管内皮细胞的受损情况及发生机理。实验共用家兔３３只，缺血组９只，其循环内皮细胞比假手术组增多（Ｐ＜０．０５）。再灌流组９只，其循环内皮细胞显著增多（与假手术组、缺血组比较，Ｐ均＜０．００１），结果显示了再灌流血管内皮细胞损伤程度较缺血组明显，同时可看到脂质过氧化物变化与循环内皮细胞数量变化有一致性。实验同时观察到针刺对再灌流出现的上述异常改变大多有明显的改善作用     

Quantification of circulating mature endothelial cells using a whole blood four-color flow cytometric assay

Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31(+), CD146(+), CD45(-), 7-amino-actinomycin-D (7AAD)(-) events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 10(6)) to ensure statistical analysis, and (4) use of 10 microm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r(2)=0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r(2)=0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0-15) in healthy individuals and 15.0/ml (range, 0-179) in patients with metastatic carcinoma (p<0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients.     

The effects of transmural pressure on prostacyclin release from porcine endocardial endothelial cells – comparison with vascular endothelial cells

 We assessed the effects of pressure on the release of prostacyclin (PGI₂) from cultured endocardial endothelial cells (EECs) and vascular endothelial cells (VECs). EECs were harvested from the right ventricle (RV) and left ventricle (LV) of porcine hearts, and VECs from pulmonary artery (PA), aorta (Ao) and coronary artery (CA). Confluent EECs and VECs were incubated for 30 min under various pressures (0, 50, 100, 150 mmHg) and PGI₂ release from each cell was measured. Pressure-induced PGI₂ release from LV-EECs was larger than that from RV-EECs. Pressure also increased PGI₂ release from both PA- and Ao-VECs, but not from CA-VECs. These findings suggest that endocardium can produce PGI₂ in response to pressure and PGI₂ released into the coronary blood from the ventricle may play an important role in the prevention of myocardial ischemia. Received: 13 August 1996 / Received after revision: 13 December 1996 / Accepted: 17 January 1997     

Circulating endothelial cells are increased in chronic myeloid leukemia blast crisis

We measured circulating endothelial precursor cells (EPCs), activated circulating endothelial cells (aCECs), and mature circulating endothelial cells (mCECs) using four-color multiparametric flow cytometry in the peripheral blood of 84 chronic myeloid leukemia (CML) patients and 65 healthy controls; and vascular endothelial growth factor (VEGF) by quantitative real-time PCR in 50 CML patients and 32 healthy controls. Because of an increase in mCECs, the median percentage of CECs in CML blast crisis (0.0146%) was significantly higher than in healthy subjects (0.0059%, P<0.01) and in the accelerated phase (0.0059%, P=0.01). There were no significant differences in the percentages of CECs in chronic- or active-phase patients and healthy subjects (P>0.05). In addition, VEGF gene expression was significantly higher in all phases of CML: 0.245 in blast crisis, 0.320 in the active phase, and 0.330 in chronic phase patients than it was in healthy subjects (0.145). In conclusion, CML in blast crisis had increased levels of CECs and VEGF gene expression, which may serve as markers of disease progression and may become targets for the management of CML.     

Antibody-mediated cellular cytotoxicity against Raji cells ADCC(RAJI): Evaluation of false positives in the detection of circulating immune complexes by Raji-cell assay

Raji-cell radioimmunoassay is a very sensitive and reproducible method for the detection of circulating immune complexes. Using a complement-independent, antibody-dependent cellular cytotoxicity assay against⁵¹Cr-labeled Raji cells, there is no correlation between activity against Raji cells and positivity in Raji-cell radioimmunoassay for circulating immune complexes in three sets of sera (from renal transplantation patients, multiparous women, and patients suffering from systemic lupus erythematosus). We conclude that IgG antibodies to Raji membranes are not a significant cause of false-positive results in circulating immune complexes as detected by Raji-cell radioimmunoassay.     

急性早幼粒细胞患者治疗前后循环内皮细胞的变化及其影响因素的分析

目的:探讨急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)患者治疗前后循环内皮细胞(circulating endothelial cells,CECs)数量、表面抗原及生物学特性的变化,并分析其与临床指标的关系。方法:免疫磁珠结合三色流式细胞仪分选计数APL患者初诊及治疗后CECs的数量;免疫荧光染色检测APL患者初诊及治疗后CECs表面CD146、CD31、CD144、VEGFR-2、CD45及CD133的表达水平。体外培养CECs进行血管生成实验及细胞集落形成率的测定。结果:APL患者CECs在外周血白细胞10×10~9/L及CD34阳性患者中数量明显增多(P0.05),高危、低危及中危3组患者的CECs数量也呈现明显的差异(P0.05)。32例APL患者诱导化疗后均获得完全缓解,治疗后CECs数量及其表面CD133表达水平均明显下降(P0.05)。同时治疗后APL患者CECs体外形成血管数目及细胞的集落形成率均明显减少(P0.05)。APL患者CECs数量治疗后/治疗前的比值与As_2O_3治疗1周后的尿砷浓度呈现明显负相关性(P0.05)。结论:精确计数并深入了解CECs生物学特性可能有助于评价APL的预后及设计治疗策略。