SD大鼠骨髓间充质干细胞体外原代培养及鉴定

目的：探讨骨髓间充质干细胞（BMSCs）体外分离培养以及扩增的方法并鉴定。方法：取100g左右雄性SD大鼠后肢股骨、胫骨骨髓,原代全骨髓培养法,多次传代纯化,体外扩增后,观察细胞形态,并免疫组化及流式细胞仪检测cd34、cd90、cd105细胞因子,鉴定是否为BMSCs。结果：所获取的细胞呈长梭形,呈现特征性的漩涡状生长,CD34阴性,CD90、CD105阳性。结论：利用全骨髓培养法成功分离骨髓间充质干细胞,10代以内的细胞纯度高,活性好。全骨髓培养较为简便、易行。     

SD大鼠骨髓间充质干细胞体外原代培养及鉴定

目的:探讨骨髓间充质干细胞(BMSCs)体外分离培养以及扩增的方法并鉴定。方法:取100g左右雄性SD大鼠后肢股骨、胫骨骨髓,原代全骨髓培养法,多次传代纯化,体外扩增后,观察细胞形态,并免疫组化及流式细胞仪检测cd34、cd90、cd105细胞因子,鉴定是否为BMSCs。结果:所获取的细胞呈长梭形,呈现特征性的漩涡状生长,CD34阴性,CD90、CD105阳性。结论:利用全骨髓培养法成功分离骨髓间充质干细胞,10代以内的细胞纯度高,活性好。全骨髓培养较为简便、易行。     

兔骨髓间充质干细胞的分离、培养与鉴定

目的:寻求家兔骨髓间充质干细胞(Bone Mesenchymal Stem cells,BMSCs)培养与分离简单易行的方法,为下一步骨髓间充质干细胞在呼吸系统疾病的应用打好基础。方法:取3个月龄家兔1只,经双侧髂前上棘抽取骨髓共5.0 ml,密度梯度离心法和贴壁筛选法相结合分离、纯化获得BMSCs,倒置差显微镜观察其形态学特性,流式细胞仪鉴定CD34、CD133表达。结果:接种细胞于24小时有少量细胞贴壁,72小时多数细胞可贴壁,贴壁细胞形态多为长梭型或多边形,原代细胞呈集落生长,12-16天可达90%融合,1%胰酶消化后传代培养,培养至第三代备用。流式细胞仪鉴定90%为骨髓间充质干细胞。结论:通过密度梯度离心法与贴壁筛选法可成功分离培养出骨髓间充质干细胞,此方法简单易行,成功率比较高。     

阳离子脂质体介导的基因转染大鼠BM-EPC的研究

目的:探讨阳离子脂质体法对体外分离培养的Sprague-Dawley(SD )大鼠骨髓来源-血管内皮祖细胞(Bone Marrow-Endothelial progenitorcells,BM-EPCs) 进行基因转染时脂质 体和DNA质粒安全有效的剂量组合.方法:体外分离、培养SD大鼠BM-EPCs ;免疫荧光技术测 定CD34、CD133、Dil-acLDL,对细胞进行鉴定;按照正交设计,用不同水平的脂质体和质粒 pEGFP转染细胞;荧光显微镜下计数阳性转染细胞,计算转染率.结果:SD 大鼠BM-EPCs细胞 表面抗原CD133、CD34呈阳性表达并具有吞噬Dil-acLDL的功能,形态学上两种细胞亚型-早 期及晚期外生EPCs共存于其中.Lipofectamine 2000和pEFGP不同剂量组合均可转染大鼠BM -EPCs,其中脂质体5ìl/质粒8ìg时的转染效率高于二者其它剂量组合(P＜0.05). 结论:优化条件后的阳离子脂质体Lipofectamine 2000可安全、有效转染体外分离培养的SD大鼠BM-EPCs.     

鼠骨髓源性内皮祖细胞的分离培养与鉴定

目的探讨大鼠骨髓源性内皮祖细胞(endothelial progenitor cells,EPCs)的分离培养鉴定的方法.方法 Percoll(1.077 g/ml)分离液分离大鼠骨髓单个核细胞,血管内皮生长因子(Vascular Endothelial Growth Factors, VEGF)和碱性成纤维细胞生长因子(basic Fibroblast Growth Factors, bFGF),对其进行诱导培养,光镜观察EPCs形态,免疫荧光检测血小板内皮细胞粘附分子-1(PECAM-1/CD31)、血管内皮钙粘蛋白(VE-cadherin/CD144)、荆豆凝集素-1(FITC-UEA-1)的表达和摄取Dil荧光标记的乙酰化-低密度脂蛋白(Dil-ac-LDL).结果 诱导培养7 d后,可见集落和铺路石样结构,激光扫描共聚焦显微镜(Laser Scanning Confocal Microscope, LSCM)显示表型为CD31+VE-cadherin+双阳性细胞以及具有内皮细胞功能的Dil-ac-LDL和FITC-UEA-1双染色细胞.结论 采用Percoll(1.077 g/ml)密度梯度离心结合VEGF、bFGF诱导培养可以获得EPCs,说明该培养方法可行.     

电针通过PI3K/AKT信号通路动员脑缺血/再灌注大鼠骨髓内皮祖细胞至外周血

目的探讨PI3K/AKT信号通路在电针动员SD大鼠骨髓内皮祖细胞(endothelial progenitor cells, EPCs)至外周血的作用研究。方法采用线栓法制备SD大鼠局灶脑缺血/再灌注模型,以"百会"穴(GV 20)及左侧"四关"穴(合谷LI4/太冲LR 3)为治疗穴位。腹腔注射PI3K特异性抑制剂LY294002阻断PI3K/AKT通路。192只雄性SD大鼠随机分为假手术组(Sham)、缺血组(I/R)、缺血+电针组(I/RE)及缺血+电针+LY294002组(I/REL)。脑缺血90min,根据再灌注时间,将各组分为再灌注24h、48h和7d三个亚组。采用流式细胞术检测骨髓和外周血CD34+EPCs数量,ELISA法检测各组大鼠骨髓AKT、磷酸化AKT(p-AKT)及内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)蛋白含量,免疫荧光共聚焦检测各组大鼠大脑皮质区CD34+微血管密度。结果脑缺血再灌注后24h和48h,大鼠骨髓和外周血CD34+EPCs数量较Sham组明显增多,EA组骨髓和外周血CD34+EPCs数量在各个观察时间点均较I/R组明显增多,I/REL组与I/RE组比较,骨髓及外周血CD34+EPCs数量无明显增高。I/R组大鼠骨髓p-AKT和eNO含量在再灌注后各时间点均较Sham组明显增多,I/RE组p-AKT及eNOS较I/R组明显增多,I/REL组与I/RE组比较,上述蛋白表达明显减少。所有组于各观察时间点总AKT蛋白表达无明显差异。此外,脑缺血再灌注后各时间点,大鼠大脑皮质缺血区CD34+MVD较Sham组明显增多。EA组CD34+微血管密度较I/R组进一步增加,I/REL组与I/RE组比较,CD34+微血管密度明显减少。结论电针可通过进一步激活骨髓PI3K/AKT信号通路动员局灶脑缺血/再灌注大鼠骨髓EPCs至外周血,促进脑血管再生。     

阿尔巴斯绒山羊骨髓间充质干细胞体外分离培养及鉴定

探讨阿尔巴斯绒山羊骨髓间充质干细胞(Arbas cashmere goat bone marrow-derived mesenchymal stem cells,g BMMSCs)体外分离培养,并对其生物学特性进行系统的鉴定,进一步深入分析体外诱导时多向分化相关基因动态表达情况。结果显示,原代培养的g BM-MSCs呈梭形或纺锤形、放射状排列的细胞集落,能够连续稳定传代至15代以上;免疫荧光及流式细胞术检测显示,g BM-MSCs表达间质系特异性表面标志物,如:CD13、CD29、CD44、CD106、CD90及CD166,不表达造血干细胞表面标志物CD45及CD34;体外诱导实验证实该细胞具有多向分化潜能,能够向脂肪细胞、成骨细胞和软骨细胞分化;荧光定量PCR实验结果显示,g BM-MSCs多向分化相关基因的表达水平随着诱导天数的增加呈上升趋势。实验结果证实阿尔巴斯绒山羊骨髓中分离培养的细胞具备g BM-MSCs的生物学特性。     

大鼠骨髓基质细胞的培养及生物学特性鉴定

目的:分离、培养大鼠MSCs,对其生物学特性进行鉴定.方法:取大鼠长骨中的骨髓组织,以贴壁法培养分离大鼠MSCs,经多次传代得到较纯的MSCs,倒置相差显微镜下观察细胞形态,应用免疫细胞化学方法对细胞的表面抗原标志进行检测.取3代MSCs,B-ME诱导6小时候后,倒置相差显微镜下观察细胞形态,应用免疫细胞化学方法鉴定诱导后细胞的表型特征.结果:倒置相差显微镜观察发现,接种后24h,细胞开始贴壁.培养3代以后,细胞呈梭形或多角形,折光性好,核为圆形、居中.免疫细胞化学检测细胞表面抗原显示:CD34-、CD45-、CD29+、CD44+、CD90+.诱导后细胞发出突起,逐渐生长延伸并彼此相连,形态学上具有神经细胞的明显特征.免疫细胞化学显示诱导后细胞NSE(神经元特异性烯醇化酶)阳性.结论:所采用的细胞分离培养方法简便可行,所获得的细胞其表型特征与文献报道一致,且具有向神经细胞分化的潜能.     

实验兔骨髓间充质干细胞的分离、培养及鉴定

探讨建立兔骨髓间质干细胞(BMSCs)的体外分离纯化、扩增和鉴定的方法,为BMSCs的进一步诱导分化和应用奠定基础。首先抽取兔髂骨骨髓,采用Percoll密度梯度离心法得到骨髓单个核细胞,接种后形成单层贴壁细胞,经胰蛋白酶消化后传代培养扩增,倒置相差显微镜观察细胞生长状态,细胞免疫组化检测CD73、CD34。结果表明,成功建立了兔BMSCs体外分离及培养扩增的方法,发现BMSCs表现贴壁生长,P0代时呈集落生长,细胞呈梭形,传代后细胞增殖速度加快,形态开始多样化;细胞免疫组化显示BMSCs表达CD73,未表达CD34,反复传代后CD73表达效率增高。上述研究表明,Percoll密度梯度离心联合骨髓贴壁法,能有效分离、纯化和扩增BMSCs,提取的细胞具有BMSCs的生长特性和抗原表型,其中P3~P6代细胞增殖能力强,可用于进一步的研究工作。     

大鼠脂肪干细胞向雪旺细胞诱导分化能力的研究

目的：研究脂肪干细胞（ADSCs）向雪旺细胞的诱导分化,为神经组织工程提供新的种子细胞。方法：取SD大鼠项背处的皮下脂肪,分离出脂肪干细胞并培养传代,流式细胞仪检测细胞表面特异标记CD29,CD34,CD44,CD45,CD90,以评价干细胞的生物学特性;采用b-FGF和forskolin等诱导脂肪干细胞向雪旺细胞分化,光镜观察诱导后细胞形态的变化;免疫荧光染色鉴定雪旺细胞特异性标记物S100、P75和GFAP的表达;PCR检测诱导前后雪旺细胞特异性标记物S100、P75的表达。结果：分离培养的鼠脂肪干细胞CD29、CB90表达呈阳性,而CD34、CD44和CD45表达呈阴性,具有脂肪干细胞的生物学特性;脂肪干细胞经过胶质细胞生长因子的作用,光镜下发现诱导的细胞形态与雪旺细胞相似;免疫荧光染色S100、P75和GFAP阳性;RT-PCR结果显示诱导的雪旺细胞标记物S100和P75表达上调。结论：脂肪干细胞可诱导分化成雪旺细胞,其表型和分子特征与雪旺细胞相似,诱导分化的脂肪干细胞是一种理想的神经组织工程的种子细胞。     

The characteristics of bone marrow-derived endothelial progenitor cells and their effect on glioma

ABSTRACT: BACKGROUND: EPCs were isolated primarily in 1997 by Asahara et al and recent studies indicate that bone-marrow-derived EPCs contribute little to the endothelium of tumor vessels. Tumors of the CNS system demonstrate various features of angiogenesis. METHODS: EPCs derived from rat bone marrow were isolated and cultured in M199 medium without any induced factors. EPCs derived from rat bone marrow were studied using immunohistochemical staining, Flow cytometry and culture under three-dimensional condition to determine EPCs' characteristics in vitro. We also established an animal model by injecting EPCs marked with Hoechst 33342 into the back of BALB/c nude mouse and performed hematoxylin-eosin (HE) and immunofluorescent staining to study EPCs' features in vivo. To research effect of EPCs on glioma, animals bearing tumors model with C6 glioma were established. About 27day after injected, we performed immunohistochemical staining and Immunofluorescence staining. RESULTS: Our results showed that EPCs derived from rat bone marrow appeared typical morphological characteristics and were positive of CD34, CD133, KDR and CD31 antigens at different time in vitro under the special M199 medium without any induced factors. The percentage of cells that expressed CD133 decreased gradually. In brief, the present study showed that EPCs derived from rat bone marrow differentiated into ECs in medium 199 without any induced factors and formed tubular structures in three-dimensional circumstances. Animal experiments suggested that EPCs differentiated into ECs and other else non-endothelial cells, and that EPCs contributed to growth of glioma. DISCUSSION: These findings provides some novel findings about biological characteristics of EPCs in vivo and ex vivo, and an update on the effect of EPCs on glioma and which would be helpful for the overall understanding of EPCs and make EPCs to be implied on the clinical therapy.     

Nonadhesive populations in cultures of mesenchymal stromal cells from hematopoietic organs in mouse and rat

The study of adhesive properties of multipotent mesenchymal stromal cells evaluated from fibroblast colony-forming units in the bone marrow of adult mice and rats in populations of cells attached and unattached to plastic substrate after 2 h to 7 days in culture demonstrated both similarities and differences. The increase in the fibroblast colony-forming units in the adhesive population peaked on day 7 of in vitro culture in both cases; however, nearly no fibroblast colony-forming units were observed in the nonadhesive population from the mouse bone marrow in this period. Conversely, the number of colonies from the rat bone marrow nonadhesive population on day 7 of culture considerably increased, and this nonadhesive population in long-term culture became the source for subsequent nonadhesive subpopulations containing fibroblast colony-forming units. After 7 days of in vitro culture, the suspension of cells isolated from the liver of 17-day-old rat fetuses also contained a fraction of unattached fibroblast colony-forming units. In the nonadhesive subpopulations from the bone marrow and fetal liver, fibroblast colony-forming units were observed up to day 48 and 30, respectively. Stromal cell precursors of nonadhesive subpopulations from the rat bone marrow featured a period of colony formation reduced to 7 days (i.e., they were formed 1.5-2 times faster compared to the primary culture). The total number of fibroblast colony-forming units from all nonadhesive subpopulations was roughly 6 and 7.4 times that of the adhesive population of the primary culture from the bone marrow and fetal liver, respectively. Considering that the mammalian bone marrow remains the preferred source of mesenchymal stromal cells, using nonadhesive subpopulations in the presented culture system can considerably increase the yield of stromal precursor cells     

The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions

Endothelial progenitor cells (EPCs) derived from bone marrow are known to be heterogeneous. In this study, we tried to find favorable conditions that induce the differentiation of mononuclear cells (MNCs) from bone marrow into EPCs. The differentiation capacity of MNCs from rat bone marrow was investigated in different conditions, such as different media, different induction times and different culture surfaces. The cell morphology and endothelial biomarkers associated with differentiated MNCs were studied. Our results indicated that MNCs cultured in EGM-2MV (Endothelial cell basal medium-2, plus SingleQuots of growth supplements) developed a bursiform shape, a late EPC-like morphology, while MNCs cultured in complete medium (CM, M199 with 10% FBS, 20 ng/mL VEGF and 10 ng/mL bFGF) showed a spindle shape, an early EPC-like morphology. Cells of both morphologies were able to incorporate DiI-ac-LDL and bind lectin in vitro. MNCs cultured in EGM-2MV exhibited a higher proliferation rate and higher eNOS expression than MNCs cultured in CM. MNCs cultured in EGM-2MV had the ability to form tubes on Matrigel. Flow cytometry results indicated that CD133 expression was highest at day 12 and that the greatest number of cells positive for both FLK-1 and CD133 appeared at day 20 from cells cultured in dishes without fibronectin coating. In addition, the expression levels of CD133, CD31 and FLK-1/CD133 were not significantly different between cells of different shapes. Our experiments suggest that MNCs from bone marrow can be differentiated into late EP-like cells in EGM-2MV, which have the ability to rapidly proliferate. These MNCs can also be differentiated into early EP-like cells in CM. Additionally, fibronectin may not be necessary for the differentiation of EPCs to mature ECs after three generations. Differentiated MNCs from bone marrow in EGM-2MV have the characteristics of EPCs, although the expression levels of EPC markers were lower than previously reported.     

两种不同内皮祖细胞的分离培养与鉴定

目的:探讨从小鼠骨髓中分离、培养、诱导分化及鉴定两种内皮祖细胞的方法,为进一步研究和临床应用奠定基础。方法:密度梯度离心法分离小鼠骨髓单个核细胞,接种于内皮祖细胞条件培养基,通过贴壁培养法培养出早期内皮祖细胞和晚期内皮祖细胞,并在0 d、6 d、10 d流式鉴定早期内皮祖细胞,在第8周流式鉴定晚期内皮祖细胞。结果:通过体外贴壁扩增培养,从小鼠骨髓细胞中成功培养出EEPC(早期内皮祖细胞)和EOC(晚期内皮祖细胞),表达CD34+/CD133+/VEGFR2+的EEPC比例从最初的0.08%能够增长至70%;EOC大约出现于3-4周,5-8周时呈现指数增长,具有典型的内皮细胞鹅卵石样形态,表达CD31、VEGFR2等内皮细胞表面标志而不表达CD34、CD133等干细胞表面标志。结论:确立了内皮祖细胞体外分离培养和诱导分化的实验方法,为进一步研究奠定基础。     

The involvement of endothelial progenitor cells in tumor angiogenesis

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood CD34, VEGFR-2, or AC 133 (CD133) antigen-positive cells, which may home to site of neovascularization and differentiate into endothelial cells in situ. Endothelial cells contribute to tumor angiogenesis, and can originate from sprouting or co-option of neighbouring pre-existing vessels. Emerging evidence indicate that bone marrow-derived circulating EPCs can contribute to tumor angiogenesis and growth of certain tumors. This review article will summarize the literature data concerning this new role played by EPCs in tumor angiogenesis.     

Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance

Bone marrow and peripheral blood of adults contain a special sub-type of progenitor cells which are able to differentiate into mature endothelial cells, thus contributing to re-endothelialization and neo-vascularization. These angiogenic cells have properties of embryonal angioblasts and were termed endothelial progenitor cells (EPCs). In general, three surface markers (CD133, CD34 and the vascular endothelial growth factor receptor-2) characterize the early functional angioblast, located predominantly in the bone marrow. Later, when migrating to the systemic circulation EPCs gradually lose their progenitor properties and start to express endothelial marker like VE-cadherin, endothelial nitric oxide synthase and von Willebrand factor. The number of circulating EPCs in healthy subjects is rather low and a variety of conditions or factors may further influence this number. In the context of possible therapeutic application of EPCs recent clinical studies employing these cells for neo-vascularization of ischemic organs have just been published. However, the specificity of the observed positive clinical effects, the mechanisms regulating the differentiation of EPCs and their homing to sites of injured tissue remain partially unknown at present.     

猫骨髓间充质干细胞的体外分离培养、纯化、鉴定

目的:分离、培养、纯化家猫的骨髓间充质干细胞,并对获得细胞的表面标志物进行鉴定,为进一步利用骨髓间充质干细胞的细胞移植实验奠定基础。方法:采用全骨髓贴壁法体外分离、培养、纯化家猫骨髓间充质干细胞,通过多次更换培养液获得较纯化的骨髓间充质干细胞,倒置相差显微镜下对细胞形态进行观察;根据第1、3、5、7、9代细胞的镜下增殖情况绘制出生长曲线;通过流式细胞仪检测细胞表面标志抗原CD34、CD44和CD90的表达率。结果:在倒置相差显微镜下观察,分离培养的骨髓间充质干细胞贴壁呈梭形或纺锤形;原代细胞生长丛集成片,5~7 d达到融合,进行传代;培养到第三代以后,细胞出现相对均匀的梭形扁平外观,迅速增殖的细胞呈涡流样排列;第3、5代骨髓间充质干细胞增殖能力强于第7、9代;采用流式细胞仪分析结果显示细胞的CD34、CD44和CD90阳性率分别为17.5%、97.9%和91%,这与骨髓间充质干细胞表面抗原的表达一致。结论:分离培养的细胞具有骨髓间充质干细胞特性,成分相对单一,第3、5代细胞纯度高,增殖能力强,适用于进一步的实验研究。     

AMD3100对apoE-/-小鼠骨髓源性内皮祖细胞增殖、迁移和黏附的影响

探讨AMD3100对apoE-/-小鼠骨髓内皮祖细胞的动员作用及其增殖、迁移和黏附的影响.12只8周龄雄性apoE-/-小鼠随机分为AMD3100组(2.5 mg/(kg·2d))和对照组(PBS 0.1 ml/2d),高脂高胆固醇饲料喂养12周后,差速贴壁法结合微孔法分离培养小鼠骨髓细胞,免疫荧光鉴定CD133/VEGFR-2双阳性细胞为内皮祖细胞;MTT比色法、Transwell、黏附试验分别检测细胞的增殖、迁移和黏附能力;通过计数典型内皮祖细胞克隆形成单位,观察次级集落单位的大小及细胞密度,检测各组内皮祖细胞的克隆形成能力;RT-PCR和Western blot检测内皮祖细胞上CXCR4 mRNA和蛋白质表达水平.与对照组比较,AMD3100组骨髓源性内皮祖细胞的增殖、迁移、黏附和克隆形成能力均显著低于对照组,其CXCR4mRNA和蛋白质表达均显著低于对照组.结果表明:持续注射AMD3100可抑制骨髓源内皮祖细胞的增殖、迁移、黏附和克隆形成能力,并下调CXCR4的表达.     

Calcitonin gene-related peptide promotes proliferation and inhibits apoptosis in endothelial progenitor cells via inhibiting MAPK signaling

Background

Calcitonin gene-related peptide (CGRP) contributes to bone formation by stimulating bone marrow stromal cell (BMSC) proliferation and differentiation. However, the proliferative and apoptotic effects of CGRP on bone marrow-derived endothelial progenitor cells (EPCs) have not been investigated.

Methods

We tested the effects of CGRP on EPC proliferation and apoptosis by Cell Counting Kit-8, flow cytometry, and studied the effects of CGRP on the expression of proliferation- and apoptosis-related markers in EPCs and the underlying mitogen-activated protein kinase (MAPK) signalling pathway by quantitative polymerase chain reaction and western blotting.

Results

We detected EPC markers (CD34, CD133 and VEGFR-2) in 7-day cultures and found that CGRP (10^??10–10^??12 M) promoted the proliferation of cultured EPCs, with a peak increase of 30% at 10^??10 M CGRP. CGRP also upregulated the expression of proliferation-associated genes, including cyclin D1 and cyclin E, and increased the percentages of G2/M-phase and S-phase cells after incubation 72 h. CGRP inhibited serum deprivation (SD)-induced apoptosis in EPCs after 24 and 48 h and downregulated the expression of apoptosis-related genes, including caspase-3, caspase-8, caspase-9 and Bax. Phosphorylated (p-)ERK1/2, p-p38 and p-JNK protein levels in EPCs treated with CGRP were significantly lower than those in untreated EPCs. Pre-treatment with the calcitonin receptor-like receptor (CRLR) antagonist CGRP8–37 or a MAPK pathway inhibitor (PD98059, SB203580 or SP600125) completely or partially reversed the pro-proliferative and anti-apoptotic effects and the reduced p-ERK1/2, p-p38 and p-JNK expression induced by CGRP.

Conclusion

Our results show that CGRP exerts pro-proliferative and anti-apoptotic effects on EPCs and may act by inhibiting MAPK pathways.

     

CXCR2-Dependent Endothelial Progenitor Cell Mobilization in Pancreatic Cancer Growth

Neovascularization is essential for tumor growth. We have previously reported that the chemokine receptor CXCR2 is an important regulator in tumor angiogenesis. Here we report that the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs) is impaired in CXCR2 knockout mice harboring pancreatic cancers. The circulating levels of EPCs (positive for CD34, CD117, CD133, or CD146) are decreased in the bone marrow and/or blood of tumor-bearing CXCR2 knockout mice. CXCR2 gene knockout reduced BM-derived EPC proliferation, differentiation, and vasculogenesis in vitro. EPCs double positive for CD34 and CD133 increased tumor angiogenesis and pancreatic cancer growth in vivo. In addition, CD133(+) and CD146(+) EPCs in human pancreatic cancer are increased compared with normal pancreas tissue. These findings indicate a role of BM-derived EPC in pancreatic cancer growth and provide a cellular mechanism for CXCR2 mediated tumor neovascularization.