C反应蛋白对内皮祖细胞部分生物学功能的影响及其机制的实验研究

目的 观察C反应蛋白(CRP)对体外培养骨髓源性内皮祖细胞(EPC)数量及增殖、迁移、黏附功能的影响及其机制探讨. 方法 密度梯度离心法获取骨髓单个核细胞,FITC-荆豆凝集紊I、DiI-乙酰化低密度脂蛋白荧光双染鉴定.单个核细胞培养7 d后进行实验分组,分为对照组和CRP干预组.CRP干预组加入不同浓度CRP(分别为5、10、15、20 μg/ml)培养48 h,然后分别采用四氮唑溴盐比色法、改良的Boyden小室和黏附能力测定来观察EPC的增殖、迁移和黏附能力.RT-PCR检测不同浓度下CRP对EPC内皮型一氧化氮合酶(eNOS)mRNA的影响,并检测EPC的NOS活性. 结果 CRP(分别为5、10、15、20 μg/ml)各组EPC数量分别为(58±3)、(54±3)、(47±3)和(39±5)个,对照组EPC数量为(60±3)个.MTT法检测CRP各亚组EPC在490 nm吸光度值分别为0.332±0.003、0.297±0.036、0.273±0.013和0.259±0.035,对照组为0.345±0.014.CRP浓度为10、15、20μ/ml 3个亚组EPC的数量及增殖能力显著低(P＜0.01).CRP各组(10、15、20 μg/ml)与对照组相比EPC黏附数量明显低[(28±2)、(22±3)、(19±3)和(16±2)个比(30±2)个,P＜0.05].不同浓度CRP各组与对照组相比EPC迁移数量明显低[(11±2)、(9±2)、(6±2)和(5±1)个比(12±2)个,P＜0.05].CRP各亚组(5、10、15、20μg/ml)EPC eNOS mRNA相对光密度显著低于对照组.CRP呈剂量依赖性降低EPC NOS活性,CRP各组EPC的NOS活性分别为(66.29±1.81)、(57.44±3.25)、(48.37±3.86)和(36.82±4.89)nmol/mg蛋白,对照组EPC NOS活性为(68.56±2.82)nmol/mg蛋白,其中浓度为10、15、20μg/ml CRP组与对照组比较差异有统计学意义(P＜0.01). 结论 CRP可能通过影响EPC eNOS表达活性降低EPC数量并影响其部分生物学功能.     

Modulation of bone marrow-derived endothelial progenitor cell activity by protein tyrosine phosphatases

Adult bone marrow contains stem cells capable of reconstituting the vascular system. The ordered progression of stem cells and more differentiated endothelial precursor cells through successive developmental stages is tightly controlled. The specialized microenvironment of the bone marrow as well as cell-autonomous processes directs the renewal and differentiation of stem cells into endothelial cells. Tyrosine phosphorylation of receptors, adaptors, and structural proteins is one mechanism whereby endothelial cell development is regulated, which involves the opposing action of protein tyrosine kinases and phosphatases. The present review focuses on the role of four nontransmembrane protein tyrosine phosphatases (TC-PTP, PTP1B, SHP-1, and SHP-2) in the self-renewal, differentiation, mobilization, and homing of endothelial progenitor cells, as well as their ability to incorporate into nascent blood vessels. Endothelial progenitor cells are known to promote vasculogenesis, accelerating restoration of blood flow to ischemic tissues, and improve cardiac function after infarct. The use of protein tyrosine phosphatase inhibitors to modulate the development and function of endothelial progenitor cells as a potential novel therapy for peripheral vascular and coronary artery disease in humans is discussed.     

自体骨髓来源内皮祖细胞移植改善脑缺血再灌注大鼠神经功能转归

目的 探讨目体骨髓米源内皮祖细肥(endothehalprogenitor cell,EPC)移植对脑缺血大鼠神经功能转归的影响及其可能机制.方法 体外分离培养自体骨髓来源EPC并用5-溴脱氧尿嘧啶核苷(5 -bromodeoxyuridine,BrdU)标记.线栓法制作大鼠大脑中动脉闭塞(middle cerebral arteryocclusion,MCAO)模型.EPC组大鼠经颈外静脉移植自体EPC[ 106/ml·kg)],对照组注射磷酸盐缓冲液(1 ml/kg),假手术组不进行任何处理(n=15).改良神经功能缺损严重程度评分(modifiedneurological severity score,mNSS)观察大鼠神经功能变化情况.BrdU免疫组化染色评价EPC增殖和分化.三维共聚焦图像分析检测脑缺血区血管结构和密度.TUNEL染色检测缺血脑组织凋亡细胞.酶联免疫吸附法检测血浆血管内皮生长因子(vascular endothelial growth factor,VEGF)浓度.结果 EPC组mNSS评分显著低于对照组[第8天时;(6.43±0.69)分对(8.86±0.95)分;q=2.673,P=0.035;第14天时:(4.55±0.89)分对(6.73±1.06)分;q=5.360,P=0.035].EPC组BrdU阳性细胞数量显著多于对照组[(42.2±5.76)对(25.67±5.49);q=4.020,P=0.030].EPC组毛细血管直径显著小于对照组[(4.51±0.21)μm对(6.34±0.24) μm;q=3.980,P =0.003];血管密度[(212.64±8.02)/0.002 mm3对(153.60±7.21 )/0.002 mn3;q =9.670,P=0.001]和微血管总表面积[(92 013±5 132)μm3/0.002 mm3对(71 366±4 538) μm2/0.002 mm3;q=4.180,P=0.014]显著高于和大于对照组;EPC组凋亡细胞数量显著少于对照组[(36.26±6.91)对(78.34±7.21);t=-4.834,P=0.003];EPC组血浆VEGF浓度显著高于对照组[(54.91±5.71)pg/ml对(13.81±4.25)pg/ml;q=12.300,P=0.002].结论 自体EPC移植对大鼠缺血脑组织具有保护作用,可能与VEGF相关联的血管再生和神经保护有关,其在治疗缺血性脑血管病中具有重要的应用前景.     

Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization in vivo

OBJECTIVE: Our laboratory and others recently provided evidence indicating that endothelial progenitor cells (EPCs) participate in postnatal neovascularization. However, the extent to which EPCs contribute to adult neovascularization remains unclear. To address this issue, we investigated the quantitative contribution of EPCs to newly formed vascular structures in an in vivo Matrigel plug assay and corneal micropocket assay. MATERIALS AND METHODS: Lethally irradiated FVB mice were transplanted with bone marrow (BM) mononuclear cells from transgenic mice constitutively expressing beta-galactosidase (beta-gal) encoded by the lacZ gene regulated by an endothelial-specific tie-2 promoter. Reconstitution of the transplanted BM leads to the expression of lacZ in mice, which is restricted to BM cells expressing tie-2. RESULTS: Four weeks after BM transplantation (BMT), tie-2/lacZ/BMT mice were implanted with either Matrigel containing fibroblast growth factor-2 subcutaneously or with a vascular endothelial growth factor pellet into the cornea. After 7 days, the Matrigel plug or the cornea was removed and analyzed by X-gal staining or immunostaining for beta-gal. X-gal staining of the Matrigel plug identified 5.7% +/- 1.2% of endothelial cells (ECs) as cells originated from BM-derived EPCs, whereas the more sensitive technique of immunofluorescence identified 26.5% +/- 0.9% of ECs. Similarly, EPC-derived cells comprised 5.0% +/- 2.4% and 17.7% +/- 3.6% of the ECs in corneal neovascularization identified by X-gal staining and immunohistochemistry, respectively. Ki67 staining of the corneal tissue documented that the majority of EPC-derived cells were actively proliferating in situ. CONCLUSION: These findings suggest that BM-derived EPCs make a significant contribution to angiogenic growth factor-induced neovascularization that may account for up to 26% of all ECs.     

Role of bone marrow-derived lymphatic endothelial progenitor cells for lymphatic neovascularization

The lymphatic vasculature plays a pivotal role in maintaining tissue fluid homeostasis, immune surveillance, and lipid uptake in the gastrointestinal organs. Therefore, impaired function of the lymphatic vessels caused by genetic defects, infection, trauma, or surgery leads to the abnormal accrual of lymph fluid in the tissue and culminates in the swelling of affected tissues, known as lymphedema. Lymphedema causes impaired wound healing, compromised immune defense, and, in rare case, lymphangiosarcoma. Although millions of people suffer from lymphedema worldwide, no effective therapy is currently available. In addition, recent advances in cancer biology have disclosed an indispensable function of the lymphatic vessel in tumor growth and metastasis. Therefore, understanding the detailed mechanisms governing lymphatic vessel formation and function in pathophysiologic conditions is essential to prevent or treat these diseases. We review the developmental processes of the lymphatic vessels and postnatal lymphatic neovascularization, focusing on the role of recently identified bone marrow-derived podoplanin-expressing (podoplanin(+)) cells as lymphatic endothelial progenitor cells.     

糖尿病小鼠缺血诱导的骨髓内皮祖细胞动员障碍

目的 观察糖尿病动物缺血诱导的骨髓内皮祖细胞（EPC）动员是否存在障碍,以及这种障碍是否和缺血诱导的血管内皮生长因子（VEGF）释放降低有关。方法 链脲霉素40mg／kg诱导C5781／6雄鼠糖尿病,非糖尿病组给予等量缓冲液。饲养2个月后,进行左侧股动脉高位结扎离断术造成后肢缺血模型,通过红四氮唑染色法与后肢血管造影确定造模成功。于术前及术后不同时间点采血（1天,3天,n：8;5天,7天及14天,n=5）,三色流式细胞术检测两组动物外周血单个核细胞中c-Ki^＋／Sea-1^＋／flk-1^＋早期EPC比例。ELISA法测定相应时间点血浆VEGF水平。结果 基础状态下,糖尿病组循环EPC数量较非糖尿病组明显减少[（0．60±0．03）％比（0．95±0．09）％,P〈0．001],血浆VEGF水平低于试剂盒检测灵敏度。两组动物缺血诱导的骨髓早期EPC释放曲线相似,即术后1天显著增加,术后3天达峰,动员持续至2周以上。但是在EPC早期快速动员阶段（术后前3天）,糖尿病组外周血早期EPC数量较非糖尿病组明显减少[1天,（1．16±0．29）％比（1．80±0．32）％,P〈0．05;3天,（1．38±0．34）％比（2．37±0．52）％,P〈0．05]。同时组织缺血也伴随着血浆VEGF浓度的显著增高：非糖尿病组血浆VEGF水平在术后一天快速增加并达到峰值,此后渐降至相对较低水平持续两周以上;而糖尿病组术后1天血浆VEGF快速释放明显降低[（73．1±18．6）pg／ml比（128．5±44．2）Pg／ml,P〈0．05]。结论 糖尿病动物基础状态下外周血早期EPC数量减少,组织缺血诱导的骨髓EPC动员障碍,这种障碍可能与缺血诱导的VEGF释放减少有关。     

骨髓源性内皮祖细胞与内皮细胞共培养促进其向成熟内皮细胞分化的研究

目的：探讨共培养的内皮细胞对内皮祖细胞向成熟内皮细胞分化的影响。方法：分离1～2个月龄,SD大鼠股骨髓,个核细胞（MNCs）,MNCs,Dil-ac-LDL与FITC-UEA-1荧光双染鉴定内皮细胞的特性。应用贴块法培养大鼠腹主动脉的内皮细胞,vWF免疫组化染色进行鉴定。采用Transwell培养板,上、下室分别加入内皮祖细胞和内皮细胞,15%胎牛血清的NO.2 DMEM/F12培养液培养14 d,倒置相差显微镜观察培养内皮祖细胞的形态。RT-PCR检测eNOS、vWF mRNA,流式细胞术检测CD31及KDR的表达。结果：荧光双染显示培养的单个核细胞具有内皮祖细胞特性;共培养的内皮祖细胞呈短梭型、铺路石状,培养至4 w时有复杂的网状结构形成。RT-PCR检测显示,eNOS及vWF mRNA表达均较对照组显著增加（P〈0.05）。流式细胞术分析表明,共培养组的内皮祖细胞CD31及KDR的表达,也显著高于对照组（分别为P〈0.05及P〈0.01）。结论：与内皮细胞共培养可促进内皮祖细胞向成熟内皮细胞分化。     

Selective resistance of bone marrow-derived hemopoietic progenitor cells to gliotoxin.

The fungal metabolite gliotoxin at low concentrations prevents mitogen stimulation of mature lymphocytes as a result of gliotoxin-induced genomic DNA degradation. Bone marrow, on the other hand, contains a subpopulation of cells resistant to gliotoxin at similar concentrations. This population includes the hemopoietic progenitor cells that grow in vitro in response to appropriate colony-stimulating factors and cells that form colonies in the spleens of lethally irradiated recipients. Gliotoxin treatment of lymph node cell-enriched bone marrow significantly delayed the onset of graft-versus-host disease in fully allogeneic bone marrow chimeras.     

低频脉冲磁场对大鼠骨髓源内皮祖细胞增殖和体外血管化的影响

目的 观察不同强度低频脉冲磁场对体外培养大鼠骨髓源内皮祖细胞增殖、细胞周期、迁移和成血管能力的影响.方法 密度梯度离心法获得大鼠骨髓源内皮祖细胞,随机分为4组:对照组,1.0mT组,1.4mT组和1.8 mT组.除时照组外.其余各组用频率为15 Hz不同强度的方波脉冲磁场刺激大鼠骨髓源内皮祖细胞,2 h/d,持续刺激5 d.曝磁5 d后,四甲基偶氮唑盐微量酶反应比色(MTT)法检测细胞增殖情况,流式细胞仪检测细胞周期,划痕试验检测细胞迁移能力,管状结构形成试验和3维培养检测细胞成血管能力.结果 1.0mT组和1.4mT组磁场促进内皮祖细胞增殖,使其细胞周期分布发生改变,DNA合成期(S期)和合成后期(G2期)细胞比例增加,1.8 mT组磁场也能促进内皮祖细胞增殖,但对细胞周期分布影响不明显.体外培养内皮祖细胞迁移能力差,各强度磁场对其迁移能力也无明显影响.不同强度磁场均能够提高内皮祖细胞成血管能力,与对照组相比差异均有统计学意义[(14.0±1.6)比(11.0±1.6); (19.2±1.9)比(11.0±1.6); (15.4±1.1)比(11.0±1.6),P均<0.05].1.0mT和1.4 mT磁场作用强于1.8mT磁场.结论 磁场的生物学作用和磁场强度相关,1.0 mT和1.4 mT低频脉冲磁场促进大鼠骨髓源内皮祖细胞增殖和DNA合成并提高其成血管能力.     

Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSC) and neural progenitor cells (NPC) are pluripotent cells. The former can give rise to myocytes, chondrocytes, adipocytes, and osteogenic cells, while the latter can give rise to astrocytes, neurons, and oligodendrocytes. The aim of this study was to analyze and compare the antigen expression patterns of MSC and NPC. DESIGN AND METHODS: Human bone marrow-derived MSC and NPC were analyzed by flow cytometry and immunocytochemistry using a variety of unique monoclonal antibodies (57D2, W4A5, W8B2) generated in our laboratory. In addition, the expression profile of CD antigens and intracellular differentiation markers was analyzed. RESULTS. We show for the first time that CD10+, CD13+, CD61+, CD90+, CD105 (endoglin)+, CD45-, CD34-, and CD133- MSC also expressed CD109, CD140b (PDGF-RB), CD164, and CD172a (SIRPa). In addition, we found heterogeneity of MSC as demonstrated by the preferential expression of nestin and W8B2 antigen on distinct MSC subpopulations. Morphologically, these populations comprised small single cells and larger cells with polygonal appearance. NPC expressed high levels of CD56, CD90 and nestin and moderate levels of CD15, W4A5, and 57D2 antigens. In contrast, CD133 and CD172 were found only on NPC subpopulations. INTERPRETATION AND CONCLUSIONS: Our data demonstrate nestin expression in most NPC as well as in immature MSC subpopulations. MSC and NPC subpopulations can now be distinguished using our novel antibodies W8B2, 57D2, and W4A5.     

雌二醇对骨髓内皮祖细胞部分生物学功能的影响

目的观察雌二醇对体外培养骨髓来源内皮祖细胞(endothelial progenitor cells,EPCs)数量及增殖、迁移、黏附功能的影响。方法密度梯度离心法获取骨髓单个核细胞,FITC-荆豆凝集素I、DiI-乙酰化低密度脂蛋白荧光双染鉴定。单个核细胞培养4天后进行实验分组,分为对照组和雌二醇治疗组。雌二醇治疗组加入不同浓度雌二醇(分别为0.001、0.01、0.1μmol/L)培养48 h,然后分别采用四氮唑溴盐比色法、改良的Boyden小室和黏附能力测定来观察EPCs的增殖、迁移和黏附能力。结果雌二醇剂量依赖性增加EPCs数量并显著改善了EPCs的黏附、迁移和增殖能力,与对照组比较差异显著。结论雌二醇可增加培养EPCs的数量并改善EPCs部分生物学功能。     

Delta-like ligand 4-Notch signaling regulates bone marrow-derived pericyte/vascular smooth muscle cell formation

Delta-like ligand 4 (DLL4) is essential for the formation of mature vasculature. However, the role of DLL4-Notch signaling in pericyte/vascular smooth muscle cell (vSMC) development is poorly understood. We sought to determine whether DLL4-Notch signaling is involved in pericyte/vSMC formation in vitro and during vasculogenesis in vivo using 2 Ewing sarcoma mouse models. Inhibition of DLL4 with the antibody YW152F inhibited pericyte/vSMC marker expression by bone marrow (BM) cells in vitro. Conversely, transfection of 10T1/2 cells with the active domains of Notch receptors led to increased expression of pericyte/vSMC markers. Furthermore, the blood vessels of Ewing sarcoma tumors from mice treated with YW152F had reduced numbers of BM-derived pericytes/vSMCs, fewer open lumens, and were less functional than the vessels in tumors of control-treated mice. Tumor growth was also inhibited. These data demonstrate a specific role for DLL4 in the formation of BM-derived pericytes/vSMCs and indicate that DLL4 may be a novel therapeutic target for the inhibition of vasculogenesis.     

Diverse contribution of bone marrow-derived late-outgrowth endothelial progenitor cells to vascular repair under pulmonary arterial hypertension and arterial neointimal formation

AimsIt is still controversial whether bone marrow (BM)-derived endothelial progenitor cells (EPCs) can contribute to vascular repair and prevent the progression of vascular diseases. We aimed to characterize BM-derived EPC subpopulations and to evaluate their therapeutic efficacies to repair injured vascular endothelium of systemic and pulmonary arteries.Methods and resultsBM mononuclear cells of Fisher-344 rats were cultured under endothelial cell-conditions. Early EPCs appeared on days 3–6. Late-outgrowth and very late-outgrowth EPCs (LOCs and VLOCs) were defined as cells forming cobblestone colonies on days 9–14 and 17–21, respectively. Among EPC subpopulations, LOCs showed the highest angiogenic capability with enhanced proliferation potential and secretion of proangiogenic proteins. To investigate the therapeutic effects of these EPCs, Fisher-344 rats underwent wire-mediated endovascular injury in femoral artery (FA) and were concurrently injected intraperitoneally with 60 mg/kg monocrotaline (MCT). Injured rats were then treated with six injections of one of three EPCs (1 × 10⁶ per time). After 4 weeks, transplanted LOCs, but not early EPCs or VLOCs, significantly attenuated neointimal lesion formation in injured FAs. Some of CD31⁺ LOCs directly replaced the injured FA endothelium (replacement ratio: 11.7 ± 7.0%). In contrast, any EPC treatment could neither replace MCT-injured endothelium of pulmonary arterioles nor prevent the progression of pulmonary arterial hypertension (PAH). LOCs modified protectively the expression profile of angiogenic and inflammatory genes in injured FAs, but not in MCT-injured lungs.ConclusionBM-derived LOCs can contribute to vascular repair of injured systemic artery; however, even they cannot rescue injured pulmonary vasculature under MCT-induced PAH.     

Sca-1 knockout impairs myocardial and cardiac progenitor cell function

Rationale: Cardiac progenitor cells are important for maintenance of myocardial structure and function, but molecular mechanisms governing these progenitor cells remain obscure and require elucidation to enhance regenerative therapeutic approaches. Objective: To understand consequences of stem cell antigen-1 (Sca-1) deletion on functional properties of c-kit+ cardiac progenitor cells and myocardial performance using a Sca-1 knock-out/green fluorescent protein knock-in reporter mouse (ScaKI). Methods and Results: Genetic deletion of Sca-1 results in early-onset cardiac contractile deficiency as determined by echocardiography and hemodynamics as well as age-associated hypertrophy. Resident cardiac progenitor cells in ScaKI mice do not respond to pathological damage in vivo, consistent with observations of impaired growth and survival of ScaKI cardiac progenitor cells in vitro. The molecular basis of the defect in ScaKI cardiac progenitor cells is associated with increased canonical Wnt signaling pathway activation consistent with molecular characteristics of lineage commitment. Conclusions: Genetic deletion of Sca-1 causes primary cardiac defects in myocardial contractility and repair consistent with impairment of resident cardiac progenitor cell proliferative capacity associated with altered canonical Wnt signaling.     

G-CSF treatment after myocardial infarction: impact on bone marrow-derived vs cardiac progenitor cells

OBJECTIVE: Besides its classical function in the field of autologous and allogenic stem cell transplantation, granulocyte colony-stimulating factor (G-CSF) was shown to have protective effects after myocardial infarction (MI) by mobilization of bone marrow-derived progenitor cells (BMCs) and in addition by activation of multiple signaling pathways. In the present study, we focused on the impact of G-CSF on migration of BMCs and the impact on resident cardiac cells after MI. MATERIALS AND METHODS: Mice (C57BL/6J) were sublethally irradiated, and BM from green fluorescent protein (GFP)-transgenic mice was transplanted. Coronary artery ligation was performed 10 weeks later. G-CSF (100 microg/kg) was daily injected for 6 days. Subpopulations of enhanced GFP(+) cells in peripheral blood, bone marrow, and heart were characterized by flow cytometry. Growth factor expression in the heart was analyzed by quantitative real-time polymerase chain reaction. Perfusion was investigated in vivo by gated single photon emission computed tomography (SPECT). RESULTS: G-CSF-treated animals revealed a reduced migration of c-kit(+) and CXCR-4(+) BMCs associated with decreased expression levels of the corresponding growth factors, namely stem cell factor and stromal-derived factor-1 alpha in ischemic myocardium. In contrast, the number of resident cardiac Sca-1(+) cells was significantly increased. However, SPECT-perfusion showed no differences in infarct size between G-CSF-treated and control animals 6 days after MI. CONCLUSION: Our study shows that G-CSF treatment after MI reduces migration capacity of BMCs into ischemic tissue, but increases the number of resident cardiac cells. To optimize homing capacity a combination of G-CSF with other agents may optimize cytokine therapy after MI.