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.     

In vitro migratory aberrancies of mesenchymal stem cells derived from multiple myeloma patients only partially modulated by bortezomib

Recent studies indicated that bone marrow mesenchymal stem cells (BM-MSCs) derived from multiple myeloma (MM) patients were different from those of normal subjects in a variety of aspects. However, it is largely unknown whether BM-MSCs derived from MM patients display any aberrant chemotactic migration. To this aim, we compared the chemotactic migration of BM-MSCs derived from MM patients with those from normal subjects. Our results showed that BM-MSCs derived from MM patients migrated more vigorously to myeloma cell line. Furthermore, proteasome inhibitor bortezomib was showed to suppress chemotactic migration of BM-MSCs whatever their origins. However, although the chemotactic migration of BM-MSCs derived from MM patients to myeloma cell line was more significantly suppressed by bortezomib treatment, migration to SDF-1 or FBS of BM-MSCs was less compromised. Both SDF-1 and TNF-α enhanced phosphorylation of iκ-Bα in BM-MSCs. Although bortezomib significantly inhibited the iκ-Bα phosphorylation by SDF-1, it had little effect on iκ-Bα phosphorylation by TNF-α. Collectively, our results suggested that aberrant chemotactic migration of BM-MSCs derived from MM patients and the possible migration-regulatory role of bortezomib treatment.     

多发性骨髓瘤患者外周血中TCR Vβ亚家族nave T细胞水平变化

目的： 检测多发性骨髓瘤（MM）患者外周血中23个TCR Vβ亚家族的T细胞受体删除DNA环（sjTRECs）的存在特点，从而了解MM患者相应Vβ亚家族nave T细胞的近期胸腺输出情况。方法: 利用半巢式PCR分别扩增12例MM患者每5×104个外周血单个核细胞（PBMCs）中的23个Vβ亚家族sjTRECs，10例正常人外周血作为对照。结果: 在5×104个PBMCs中，检测到MM患者sjTRECs的Vβ亚家族数量约为（5.00±2.45）个，与正常人的（9.60±5.48）个相比，检出的亚家族数量明显减少（P<0.05）；23个Vβ亚家族sjTRECs在正常人中均可以检测到，而在MM患者仅检测到部分；并且Vβ2、Vβ10、Vβ16、Vβ17和Vβ21等5个亚家族sjTRECs的检出率明显低于正常水平。12例MM患者检出的亚家族数量（2-9个）不等，患者的年龄与检出的亚家族数量存在负相关（r=-0.892；P<0.01）。结论: MM患者胸腺近期输出的23个Vβ亚家族nave T细胞存在不同程度的缺失或水平降低，表明患者存在不同程度的细胞免疫功能缺陷以及T细胞谱系重建的能力和潜能受损。     

Biocompatibility of porcine small intestinal submucosa and rat endothelial progenitor cells in vitro

Objective: This study investigated the biocompatibility of the small intestinal submucosa (SIS) and endothelial progenitor cells (EPCs) by co-cultivating EPCs and SIS in vitro and observing EPC growth on the SIS. Methods: The porcine SIS was prepared and bone marrow mononuclear cells (BMMNCs) were isolated from 3 or 4-week old male SD rats. Cellular morphology was observed by light microscopy and scanning electron microscopy (SEM) and viabilities by the MTT assays. Endothelial progenitor cells (EPCs) were phenotyped by immunocytochemistry, immunofluorescence microscopy and flow cytometry. Vascular lumen formation was evaluated by the Matrigel tube formation assays. EPCs were seeded onto the SIS and production of angiogenin-1 and endothelial cell growth factor (VEGF) by EPCs was examined by ELISA and immunoblotting assays. Results: Light microscopy and SEM showed that the mechanically and chemically treated small intestinal submucosa was composed of cell-free extracellular matrix. Immunohistochemistry, and flow cytometry revealed that the EPCs expressed appropriate surface markers including CD34, CD133, and VEGFR-2. Furthermore, the EPCs formed lumen-like structures and the SIS significantly enhanced the growth of EPCs in vitro. Conclusion: SIS has good biocompatibility with EPCs. SIS pre-seeded with EPCs can be potentially applied as an alternative scaffold material in artificial blood vessel prosthesis.     

PWM-induced generation of immunoglobulin-secreting cells in patients with multiple myeloma

Summary The pokeweed-mitogen-induced transformation of B-lymphocytes into immunoglobulin-secreting cells was studied in vitro in 25 patients with multiple myeloma using a reverse hemolytic plaque assay. Fifteen patients showed a good response in generating immunoglobulin-secreting cells, whereas 10 patients showed a decreased B cell reactivity which was not due to intermittent melphalan/steroid therapy administered to 15 patients. Experiments with lymphocyte subpopulations demonstrated that the inability of some multiple-myeloma patients to generate immunoglobulin-secreting cells was always based on a defect in the B-cell subset. Co-culture experiments with lymphocytes from normal individuals and patients revealed a cell-mediated suppression in one case, whereas humoral suppressive factors in the patients' serum could not be observed using the reverse hemolytic plaque assay. Patients were classified into three groups: (a) patients with a normal B-cell function, (b) patients with a reversible, tumor-dependent suppression of B-cell reactivity and (c) patients in whom the normal B-cell population was replaced by non-reactive cells.

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Abbreviations Ig Immunoglobulin(s) - RHPA Reverse hemolytic plaque assay - ISC Immunoglobulin-secreting cells - PWM Pokeweed mitogen - FCS Fetal calf serum - MM Multiple Myeloma Supported by the Deutsche Forschungsgeminschaft (Ru 21 5/2)     

冠状动脉钙化与成骨细胞表型的内皮祖细胞相关性

目的:探讨冠状动脉钙化与表达成骨细胞表型的内皮祖细胞(EPC-OCN)含量的关系。方法:收集上海新华医院行冠状动脉CT血管造影的患者48例,以钙化积分(CACS)定量评估冠脉钙化程度。其中冠状动脉无明显或轻度钙化的记为轻、中、重度钙化组。流式细胞术分析以CD34、KDR和OCN抗体所标记外周血单个核细胞中的EPC数量及EPC-OCN含量。结果:轻度钙化组EPC数量较中度及重度钙化组明显增多;重度钙化组表达EPC-OCN比例显著高于轻、中度钙化组;3组EPC-OCN数量无显著差异;多因素回归分析显示,EPC数量与钙化积分呈负相关;而EPC-OCN比例与钙化积分、血磷浓度、空腹血糖、糖化血红蛋白呈正相关。结论:冠状动脉钙化患者循环EPC数量减少,与钙化积分呈负相关;冠状动脉严重钙化患者EPC-OCN比例升高,并与钙化积分、血磷浓度、空腹血糖和糖化血红蛋白呈正相关。     

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。     

Expanded natural killer cells augment the antimyeloma effect of daratumumab,bortezomib, and dexamethasone in a mouse model

The use of natural killer (NK) cells is a promising and safe immunotherapeutic approach in the field of cancer immunotherapy. However, combination treatments are required to enhance the effector functions and therapeutic efficacy of NK cells. In this study, we investigated the potential of daratumumab (Dara), bortezomib, and dexamethasone (Dvd) to augment the antitumor effects of NK cells in a multiple myeloma (MM) xenograft mouse model. NK cells were expanded and activated using the K562-OX40 ligand and membrane-bound IL-18 and IL-21 in the presence of IL-2 and IL-15 from peripheral blood mononuclear cells from MM patients. A human MM xenograft model was established using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγ^null (NSG) mice. Tumor-bearing mice were divided into six treatment groups: no treatment, expanded NK cells (eNKs), Dara, Dara + eNKs, Dvd, and Dvd + eNKs. Dvd treatment strongly enhanced the cytotoxicity of eNKs by upregulating expression of NK cell activation ligands, downregulating expression of NK cell inhibitory ligands, and promoting antibody-dependent cellular cytotoxicity. The combination of eNKs with Dvd significantly prolonged mouse survival and reduced the tumor burden and serum M-protein level. Furthermore, Dvd pretreatment significantly increased eNK persistence and homing to MM sites. Our findings suggest that Dvd treatment potentiates the antimyeloma effects of NK cells expanded and activated ex vivo by modulating immune responses in MM-bearing mice.     

系统性红斑狼疮患者外周血内皮祖细胞的实验研究

目的探讨系统性红斑狼疮患者外周血内皮祖细胞（EPC）数量、功能是否改变。方法连续选入33例系统性红斑狼疮（SLE）患者做为SLE组,33名门诊健康体检者为对照组。流式细胞分析计量外周血CD34和KDR双阳性细胞,同时原代培养EPC,培养7 d后MTT法检测增殖能力,改良boydon小室法检测迁移能力。结果SLE组外周血EPC数量较对照组显著减少,P〈0.01;SLE组的增殖、迁移能力均较对照组减弱,P〈0.01。结论系统性红斑狼疮患者外周血EPC数量减少,增殖、迁移功能降低,可能是SLE促动脉粥样硬化发生的机制之一。     

稳定性心绞痛患者内皮祖细胞与侧支循环之间的关系探讨

目的： 探讨稳定性心绞痛(SAP)冠状动脉有重度狭窄或完全闭塞患者循环内皮祖细胞（EPCs）与侧支循环的关系。 方法: 将2006年3月至2007年8月期间入院的稳定性心绞痛患者，经冠状动脉造影检查冠脉有重度狭窄或完全闭塞的，将其侧支循环根据Rentrop分级分为0、1、2、3级，Rentrop分级≥2级者归为侧支循环良好组（Coll＋组,n=15),≤1级者归为侧支循环不良组（Coll-组,n =15）,共2组。经造影导管抽取血样标本，分离培养单个核细胞。激光共聚焦显微镜鉴定Dil标记的乙酰化低密度脂蛋白（Dil-acLDL）和FITC标记的荆豆凝集素-1(FITC-UEA-1)双染色阳性细胞为正在分化的EPCs，并计数。采用改良的Boyden小室和MTT法分别测定其迁移能力及增殖能力。 结果: Coll+组内皮祖细胞数量(60.9±9.6)EPCs/视野(×400)显著高于Coll-组（31.0±4.1）EPCs/视野,P﹤0.01,其迁移能力和增殖能力也明显强于Coll-组（P<0.01），相关性分析显示EPCs的数量、迁移能力和增殖能力与侧支循环程度呈正相关。 结论: 本研究显示了稳定性心绞痛患者循环内皮祖细胞与侧支循环的形成之间存在着关联性。     

人脐带血中内皮样前体细胞的初步研究

目的 观察脐带血中内皮细胞的前体细胞，并研究该前体细胞分化成熟所需条件。方法 采用常规分离方法，分离脐带血中的单个核细胞，培养于含小牛下丘脑提取液和地塞米松的MCDB131培养液中，以获得具有粘附性的细胞，于倒置相差显微镜下每日观察两次一并记录。结果 初步的实验结果表明脐带血中存在内皮细胞的前体细胞，小牛下丘脑提取物对该细胞的生成有促进作用，而地塞米松对其生成有抑制作用。结论 初步证实脐带血中有内皮细胞的前体细胞存在，并对其分化发育条件有了初步认识，为对其进一步的研究及应用打下了基础。     

内皮祖细胞在糖尿病血管病变中的作用及干预治疗

血管内皮祖细胞(endothelial progenitor cells, EPCs)是一种能直接分化为血管内皮细胞的前体细胞,它与出生后的血管形成密切相关,具有重要的病理生理学意义。近年的研究显示EPCs功能受损和数量减少在糖尿病血管病变发生的机制中扮演着重要角色。从单个核细胞移植、内源性EPCs的动员活化以及EPCs的基因修饰这几个方面进行研究,为糖尿病血管病变治疗提供了新思路。本文就EPCs的生物学特性及其与糖尿病血管病变的关系以及内皮祖细胞对糖尿病血管病变治疗的展望进行综述。     

含硼替佐米方案对多发性骨髓瘤患者出凝血功能及循环中微颗粒水平的影响*

 目的：研究采用含硼替佐米方案（PAD）诱导治疗的多发性骨髓瘤（MM）患者出凝血功能及循环中总微颗粒（TMPs）水平的变化特点。方法：使用流式细胞术检测38例新诊断MM患者和30例健康志愿者TMPs水平,观察MM患者PAD治疗前后血小板、凝血、抗凝、纤溶功能和TMPs的改变。结果：治疗前MM患者FVIII:C和vWF:Rco升高［(152.89±31.14)%和(165.69±38.43)%］;血小板聚集功能下降［(63.76±21.36)%］;PAI升高［3.98(1.63)U/mL］;TMPs较健康对照组升高［（640.65±214.22）μL vs （134.29±63.09)μL,P<0.01］,并与β2-MG正相关（r=0.672,P<0.01）。PAD治疗后,血小板聚集功能恢复［(77.83±15.62)%,P=0.01］;PAI异常得到纠正［0.88(1.38)U/mL,P<0.01］;TMPs也进行性下降,3疗程PAD后降至（184.25±93.35）/μL（P<0.01）。结论：MM患者本身具有血小板、凝血、纤溶异常和TMPs水平升高;PAD方案使疾病缓解同时恢复患者血小板功能,纠正纤溶异常,并降低TMPs水平,以上改变可能是采用含硼替佐米方案治疗的MM患者血栓发生率低的部分原因。     

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。     

血管内皮祖细胞与缺血性心血管病的血管新生

1997年Ｔａｋａｙｕｋｉ等[1] 在Ｓｃｉｅｎｃｅ杂志发表论文报道血管内皮祖细胞 (ｅｎｄｏｔｈｅｌｉａｌｐｒｏｇｅｎｉｔｏｒｃｅｌｌｓ ,ＥＰＣ)分离成功 ,并可用于体内血管新生 (ａｎｇｉｏｇｅｎｅｓｉｓ)。当时提出的血管内皮祖细胞认为是假定的 (ｐｕｔａｔｉｖｅ)。但是 ,近年来随着干细胞研究的深入 ,血管内皮祖细胞的存在基本已经确认 ,而且它与血管新生间的关系日益受到重视[2 ,3 ] 。本文将讨论血管内皮祖细胞的特征 ,它与缺血性心血管疾病中血管新生的关系及其可能的应用前景。1　血管内皮祖细胞的特征　　血管内皮祖细胞是血…     

急性淋巴细胞白血病患者FGFR3表达对循环内皮细胞增殖的影响

目的:了解急性淋巴细胞白血病(ALL)患者成纤维细胞生长因子受体3(FGFR3)表达对循环内皮细胞(CECs)增殖的影响。方法:通过RT-PCR法检测44例ALL患者骨髓中FGFR3 mRNA表达,分成FGFR3~+组及FGFR3~-组进行Kaplan-Meier生存分析。利用免疫磁珠结合流式细胞术分离计数CECs,对比两组CECs数量、表面抗原表达、生长曲线及集落生成数的差异。免疫荧光组化染色测定CECs表面FGFR3表达水平。结果:44例核型正常ALL患者中FGFR3 mRNA表达阳性率为43.2%,T-ALL组的FGFR3表达高于B-ALL组(P0.05)。19 d骨髓原始细胞比例≥5%的患者FGFR3表达升高(P0.05)。FGFR3阳性组的总生存率明显低于阴性组(P0.05)。分离出的CECs高表达CD31、CD144、VEGFR-2和CD146,基本不表达CD45。与FGFR3阴性组相比,阳性组CECs数量、CD133的阳性率及集落生成数均增多(P0.05)。体外培养中,FGFR3阳性组与阴性组相比,生长曲线3个时点上的细胞数差异有统计学显著性(P0.05)。19例ALL-FGFR3~+患者CECs表面均能检测到FGFR3表达,阳性率为29.00%±15.71%。结论:致癌基因FGFR3对ALL患者CECs的增殖有促进作用,可能具备了抗肿瘤及抗血管生成的双重靶点身份,可以为今后的分子治疗提供更多的选择。     

皮肤来源间充质干细胞对内皮祖细胞募集效应的体外观察

目的细胞的募集趋化与归巢被认为是间充质干细胞（MSCs）修复重建受损组织的重要机制,本研究拟通过体外实验观察皮肤来源间充质干细胞对在血管化中发挥重要作用的内皮祖细胞（EPCs）的募集效应。方法分离与培养胎鼠皮肤MSCs和大鼠EPCs,利用免疫组织化学法,分别检测二者的SDF-1与CXCR4表达;并利用体外迁移实验,观察MSCs是否影响EPCs的迁移。结果利用免疫组织化学检测,MSCs和EPCs可以分别表达SDF-1与CXCR4;利用双室培养系统发现,MSCs可明显促进EPCs的迁移,且阻断SDF-1表达后,这种促进效应得到抑制（P〈0.05）。结论体外实验证实了MSCs可以促进EPCs的募集趋化。     

红斑狼疮患者外周血内皮祖细胞数量与颈动脉内膜-中膜厚度相关性研究

目的探讨系统性红斑狼疮（SLE）患者外周血内皮祖细胞（EPC）与颈动脉内膜-中膜厚度（IMT）的关系。方法连续选入40例SLE患者做为SLE组,超声检测颈动脉IMT按IMT增厚与否分为IMT正常组和IMT增厚组,20名门诊健康体检者为对照组。流式细胞分析计量外周血EPC（CD34和KDR双阳性细胞）;比较各组EPC数量及直线相关分析EPC数量和IMT相关性。结果SLE组外周血EPC数量较对照组显著减少,P〈0.01;同时在SLE组中IMT增厚组EPC数量低于IMT正常组,P〈0.01,且IMT与外周血EPC数量呈负相关（n=40,r=-0.494,P=0.006）。结论SLE患者存在早期动脉粥样硬化,外周血EPC数量降低与颈动脉IMT增加密切相关,外周血EPC数量降低可能是SLE引起早期动脉粥样硬化的重要原因之一。     

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.     

High immunoproteasome concentration in the plasma of patients with newly diagnosed multiple myeloma treated with bortezomib is predictive of longer OS

PurposeProteasome inhibitors (PI) bortezomib or carfilzomib among them, play a crucial role in the modern standard therapy for multiple myeloma (MM). In this study, we intended to evaluate whether immunoproteasome (IMP) concentration could act as an effective biomarker which determines the probability of response to treatment with bortezomib, in order to detect groups of patients who are more likely to respond to treatment with PI.Materials and methodsIn our study, we evaluated IMP concentration in the plasma of 40 patients with monoclonal gammopathy of undetermined significance (MGUS) and 116 patients with newly diagnosed MM during treatment with or without PI.ResultsThe values of all the studied parameters after the applied chemotherapy in the responders’ group of patients declined considerably during the consecutive cycles of chemotherapy compared to their initial levels. On the contrary, in the group of non-responders, we observed no change in the measured IMP parameters during the consecutive cycles of therapy. We also showed that higher baseline IMP concentration might indicate longer overall survival (OS) in all patients.ConclusionsOur results indicate that assessing plasma IMP concentration can be applied as a strong biomarker for predicting clinical response to treatment and OS in patients with newly diagnosed MM.