差速贴壁法培养大鼠骨髓内皮祖细胞的实验研究

目的:探索一种经济、可靠、稳定、高纯度的内皮祖细胞培养方法。方法:采用密度梯度离心法分离培养大鼠骨髓血管内皮祖细胞,于包被人纤连蛋白的25mL培养瓶中贴壁培养,收集48h后的未贴壁细胞培养至第7天,通过细胞摄取DiL-acLDL、结合FITC-UEA-1的荧光双染阳性率从功能角度鉴定细胞,并通过流式细胞术动态测定细胞表面抗原CD133、flk-1、VE-cadherin(CD144)及CD31进一步鉴定。结果:48h未贴壁细胞培养,细胞呈短梭形、多角形,胞体小;可见到大量典型的内皮祖细胞克隆;细胞摄取DiL-acLDL,结合FITC-UEA-1荧光双染阳性率为(87.0±6.0)%。细胞表面抗原CD133、flk-1、CD144和CD31阳性率分别为61.01%、8.55%、6.18%和8.20%。结论:差速贴壁法是一种经济、可靠、稳定、高纯度的内皮祖细胞培养方法。     

人外周血内皮前体细胞体外增殖规律的动态观察及其特性

目的：探讨人外周血内皮前体细胞（endothelial progenitor cells EPCs）体外诱导分化为内皮细胞（ECs）的生长增殖规律及其特性，以期证实人外周血是临床治疗缺血性疾病一个理想的内皮前体细胞来源。方法密度梯度离心提取单个核细胞，接种在人纤连蛋白包被的培养板上，培养于含有促血管生长因子VEGF、b—FGF、IGF、EGF等的内皮生长基质EGM-2 MV中。每天倒置显微镜观察，并记录。4d后去除未附着细胞，继续培养黏附细胞，同时，黏附细胞用Dn—AC—LDL和FITC—UEA—1进行免疫荧光染色，荧光显微镜和共聚焦激光扫描显微镜观察。收集第7d的黏附细胞，流式细胞仪检测细胞表面标志CD34和CD31。结果接种1d后，一些细胞变形；2d后，有细胞团形成；3d后，细胞团周围一些贴壁细胞开始出现；4d后，黏附细胞呈短梭形或多角形贴壁生长，荧光显微镜、共聚焦激光扫描显微镜下可观察到Dn—AC—LDL和FITC—UEA-1双阳性的黏附细胞；第6d、7d，黏附细胞呈长梭形；FACS分析，CD34和CD31阳性率分别为（14．13±2．79）％、（54．67±3．44）％。结论外周血中确实存在EPCs，并且在促血管生长因子VEGF、b—FGF、IGF、EGF等的刺激下能分化为成熟的内皮细胞。EPCS可望作为临床血管再生的细胞治疗     

大鼠心脏血内皮祖细胞的分离培养、鉴定与分化

目的研究大鼠心脏血来源的内皮祖细胞(EPCs)分离、体外培养、鉴定及诱导分化的方法。方法密度梯度离心法从大鼠心脏血中分离单个核细胞,接种于铺有纤维连接蛋白的培养瓶内,给予含有血管内皮生长因子(VEGF)等的M199培养基,采用免疫组织化学技术和流式细胞仪进行EPCs的细胞表面标志物检测。结果培养3～4d,可观察到梭形贴壁细胞,14d左右贴壁细胞呈条索状结构,贴壁细胞是表达血管内皮细胞的特异性标志。结论大鼠心脏血中含有EPCs,在一定条件下,可分化成血管内皮细胞。     

造血干细胞表面标志物AC133的研究进展

AC133是近几年才发现的分子质量为119 ku的新型糖蛋白,主要选择性表达于成人骨髓、胎肝、脐带血及外周血等造血组织的CD34~+造血干、祖细胞(HSPC),而不表达于血管内皮细胞。AC133抗原是继CD34之后发现的又一重要的HSPC表面标志,并且可能更优于CD34原始细胞标志,具有很好的应用前景。本文就AC133的研究进展作一综述,重点介绍其抗原结构、抗体的制备和分析、表达、与其他抗原关系及应用前景。     

血管细胞外基质胶促进骨髓CD34+祖细胞分化为内皮细胞的实验研究

目的 研究血管细胞外基质(ECM)胶促进骨髓CD34⁺祖细胞分化为内皮细胞的作用。方法 将人主动脉组织脱细胞后得到ECM，胃蛋白酶消化后得到ECM胶。HE染色和DAPI染色观察主动脉组织脱细胞效果。将小鼠骨髓CD34⁺祖细胞分为2组：纤连蛋白(FN)组置于涂有FN的培养板中培养；ECM组置于板底铺有人主动脉ECM胶的培养板中培养。逆转录聚合酶链反应(RT-PCR)检测CD34⁺祖细胞CD31、内皮型一氧化氮合酶(e NOS)及血管性血友病因子(VWF)mRNA表达；流式细胞术检测CD34⁺祖细胞表面标志物CD31、CD144、血管内皮生长因子受体2(VEGFR2)、CD133及CD45阳性细胞百分率；荧光显微镜下观察吞噬荧光素标记的乙酰化低密度脂蛋白(DiIacLDL)的细胞数；蛋白芯片检测细胞分泌的促血管生成因子含量。结果 DAPI和HE染色均显示人主动脉组织脱细胞前含有大量细胞核，而脱细胞后得到的血管ECM中不含细胞核。与FN组相比，ECM组CD34⁺祖细胞CD31、eNO...     

X染色体耦联锌指蛋白在人脑胶质瘤干祖细胞中的表达及意义

目的探讨X染色体耦联锌指蛋白(Zfx)在人脑胶质瘤干祖细胞中的表达及意义。方法逆转录PCR方法检测人脑胶质瘤干祖细胞SU2及其分化后贴壁细胞中mRNA的表达;细胞免疫组化检测人脑胶质瘤干祖细胞SU2中Zfx、CD133、nestin的表达;然后在含有10%胎牛血清培养液中诱导SU2分化,并检测分化后的细胞中Zfx、S100、GFAP的表达。结果 Zfx在胶质瘤干祖细胞SU2及其分化后贴壁细胞中的mRNA均有表达。Zfx在人脑胶质瘤干祖细胞SU2中的蛋白水平表达高,但SU2经诱导分化后细胞中Zfx蛋白水平表达减弱。结论 Zfx在人脑胶质瘤干祖细胞中高表达,可能在人脑胶质瘤干祖细胞自我更新和分化抑制过程中起重要作用。     

Syndecan-1在神经干细胞的表达及其与AC133的关系

目的 研究syndecan-1（CD138）在人的胚胎神经干细胞上的表达及其与AC133的关系。方法 采用免疫荧光标记和流式细胞仪分析。通过CD138、AC133直标单克隆抗体（mAb）观察神经干细胞细胞膜上CD138和AC133荧光标记阳性率;同时通过双重免疫荧光标记法,观察神经干细胞细胞膜上共表达CD138和AC133的百分比。结果 神经干细胞上均表达CD138和AC133分子,同时,表达AC133分子的冲经干细胞约有50％左右表达CD138分子。结论 在神经干细胞的早期已有CD138分子的表达。其对神经干细胞的生长、分化等作用值得进一步研究。     

从PC-3细胞中富集前列腺癌干细胞的实验研究

目的从PC-3人前列腺癌细胞中分离并富集前列腺癌干细胞。方法体外无血清悬浮培养PC-3细胞,通过传代更新、诱导分化验证PC-3悬浮成球细胞具有强增殖性、自我更新及分化潜能,实时荧光定量核酸扩增检测系统检测CD44和CD133mRNA的表达,流式细胞术检测CD44+CD133+细胞和侧群(SP)细胞比例。结果 PC-3成球细胞可以在无血清培养液(SFM)中生存并形成可以稳定传代的悬浮细胞球。PC-3成球细胞具有自我更新和分化能力,其前列腺癌干细胞标志物CD44和CD133mRNA的相对表达量分别是PC-3贴壁细胞的41.83倍和10.48倍(P<0.05)。PC-3悬浮成球细胞中CD44+CD133+细胞比率为13.94%,SP细胞含量为3.10%,均显著高于PC-3贴壁细胞的0.77%和0(P<0.05)。结论通过无血清悬浮聚球培养可以从PC-3细胞中简便、高效地富集前列腺癌干细胞。     

Expression and significance of X-linked zinc-finger protein in human glioma stem/progenitor cells

目的 探讨X染色体耦联锌指蛋白(Zfx)在人脑胶质瘤干祖细胞中的表达及意义.方法 逆转录PCR方法检测人脑胶质瘤干祖细胞SU2及其分化后贴壁细胞中mRNA的表达；细胞免疫组化检测人脑胶质瘤干祖细胞SU2中 Zfx、CD133、nestin的表达；然后在含有10％胎牛血清培养液中诱导SU2分化,并检测分化后的细胞中Zfx、S100、GFAP的表达.结果 Zfx在胶质瘤干祖细胞SU2及其分化后贴壁细胞中的mRNA均有表达.Zfx在人脑胶质瘤干祖细胞SU2中的蛋白水平表达高,但SU2经诱导分化后细胞中Zfx蛋白水平表达减弱.结论 Zfx在人脑胶质瘤干祖细胞中高表达,可能在人脑胶质瘤干祖细胞自我更新和分化抑制过程中起重要作用.     

血管内皮细胞生长因子-165和血管形成素-1促进干细胞动员

目的 研究缺血肌肉转染腺病毒(Ad-)携带人血管内皮细胞生长因子-165(VEGF-165)和人血管形成素-1(Ang-1)基因后引起的细胞动员效应。方法 制作大鼠下肢血管闭塞模型,肌肉内注射Ads-VEGF-165和／或Ad5-Ang-1,以Western法检测生长因子蛋白表达、免疫组化检测基因导入后在缺血肌肉引起的效应。结果(1)局部转基因肌肉组织高表达人VEGF-165蛋白和人Ang-1蛋白;(2)与对照组相比,接收两因子转染后的骨骼肌纤维间溴化脱氧尿嘧啶(BrdU)阳性的浸润细胞显著增多,呈协同效应。有细胞同时表达内皮细胞、骨骼肌细胞、平滑肌细胞标志性抗原。部分细胞表达CD117^ 并分化为新生微血管的内皮细胞。结论(1)两因子能促进CD117^ 骨髓干细胞动员到缺血组织部位并分化为内皮细胞参与血管新生;(2)两因子可能协同动员召集能在缺血部位聚集并分化为其它类型细胞的干／祖细胞;(3)两因子可能同时作为血管生成因子和细胞动员剂用于缺血性疾病的干细胞移植治疗中,以增强缺血损伤修复效果和节省干细胞用量。     

银杏叶提取物对外周血内皮祖细胞数量和功能的影响

目的观察银杏叶提取物对外周血内皮祖细胞(endothelial progenitor cell, EPC)数量和功能的影响。方法密度梯度离心法获取外周血单个核细胞，培养7 d后，收集贴壁细胞并加入银杏叶提取物(10, 25和50 mg·L^-1)干预一定时间(6,12,24和48 h)。激光共聚焦显微镜和流式细胞仪鉴定EPC，分别观察EPC的增殖、迁移、粘附和体外血管生成能力。结果银杏叶提取物促进外周血EPC扩增，25 mg·L^-1银杏叶提取物作用24 h对EPC数量的影响最为显著(较对照组增加了1倍， p<0.01)。银杏叶提取物也显著改善了外周血EPC的粘附、迁移、增殖和体外血管生成能力。结论银杏叶提取物可增加EPC数量并改善其功能。     

阻塞性睡眠呼吸暂停患者外周血内皮祖细胞及 促血管生成因子水平研究

摘要： 目的 观察阻塞性睡眠呼吸暂停 （OSA） 患者外周血内皮祖细胞 （EPC） 不同亚族和促血管生成因子水平的变化, 探讨不同程度 OSA 患者外周血 EPC 对血管修复的可能性。方法 选取 90 例 OSA 患者和 30 例健康志愿者 (对照组), 根据睡眠呼吸暂停低通气指数(AHI)将 90 例 OSA 患者均分为轻、 中、 重度 OSA 组。密度梯度离心法提取单个核细胞, 依据乙醛脱氢酶(ALDH)活性对 EPC 进行分选, 流式细胞仪联合 CD133、 CD34、 含激酶域插入片段受体 (PE-KDR)相应细胞表面标志物测定 CD133+ KDR+ EPC 及 CD133+ CD34+ EPC、 CD34+ KDR+ EPC、 ALDHlo CD34+ KDR+ EPC 的水平。酶联免疫吸附试验(ELISA)测定患者外周血低氧诱导因子-1α(HIF-1α), 血管内皮生长因子(VEGF)及基质细胞衍生因子-1α(SDF-1α)的水平。结果 对于外周血 CD133+ KDR+ EPC、 CD133+ CD34+ EPC、 CD34+ KDR+ EPC 水平, 重度 OSA 组>中度 OSA 组>轻度 OSA 组>对照组 （均 P < 0.05）; 轻、 中度 OSA 组的外周血 ALDHlo CD34+ KDR+ EPC 水平高于对照组, 重度 OSA 组低于其他 3 组 （均 P < 0.05）; 血清 HIF-1α、 VEGF 均是重度 OSA 组>中度 OSA 组>轻度 OSA 组>对照组, SDF-1α水平为重度 OSA 组<中度 OSA 组<轻度 OSA 组<对照组 （均 P < 0.05）。结论 OSA 患者可能都会诱导动员并招募大量无效 EPC, 其数量庞大, 但直接参与修复内皮的 ALDHlo CD34+ KDR+ EPC 并未增加, 尤其对于重度 OSA 患者甚至有可能减少, OSA 减弱了修复内皮的可能性, 加重了内皮损伤, 从而增加心血管事件的发生风险。     

Effects of puerarin on number and activity of endothelial progenitor cells from peripheral blood

AIM: To investigate whether puerarin can augment endothelial progenitor cells (EPCs) numbers, promote EPC proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. METHODS: EPCs were characterized as adherent cells by double staining of DiLDL-uptake and lectin binding under a laser scanning confocal microscope. Expression of KDR, VEGFR-2, and AC 133 was detected by flow cytometry. EPCs proliferation, migration and in vitro vasculogenesis were determined with MTT assay, modified Boyden chamber assay, and in vitro vasculogenesis kit, respectively. EPCs adhesive assay was performed by replating those on fibronectin-coated dishes, then adherent cells were counted. RESULTS: Incubation of isolated human MNCs with puerarin 0.1-3 mmol/L increased the number of EPCs, EPC proliferative, migratory, adhesive, and in vitro vasculogenesis capacity in a concentration and time-dependent manner, which reached maximum at 3 mmol/L, 24 h (approximately 1-fold increase, P<0.01). CONCLUSION: Puerarin enhanced     

Circulating endothelial progenitor cells are reduced in hemodialysis patients

OBJECTIVE: The risk of cardiovascular disease in hemodialysis patients is far greater than in the general population. Endothelial progenitor cells (EPCs) circulating in the peripheral blood contribute to neovascularization in the ischemic tissue. EPCs are considered to be included in CD34 positive (CD34+) or AC133 positive (AC133+) mononuclear cells (MNCs). This study's aim was to determine the number and functional activity of EPCs in hemodialysis patients and age-matched control subjects. METHODS: The numbers of CD34+ MNCs and AC133+ MNCs in the peripheral blood were quantified by flow cytometry. The peripheral blood EPCs were also examined by an in vitro culture assay. The levels of serum vascular endothelial growth factor (VEGF) were measured by sandwich enzyme immunoassay. RESULTS: The numbers of CD34+ MNCs and AC133+ MNCs were significantly reduced by 56% and 49%, respectively, in hemodialysis patients (n = 50) compared with control subjects (n = 36). The number of EPCs determined by the culture assay was also significantly reduced by 41% in hemodialysis patients compared with control subjects. Multivariate analysis revealed that none of the atherosclerotic risk factors were independent predictors of reduced CD34+ MNC counts. The serum VEGF levels in hemodialysis patients were not different from those in control subjects and did not correlate with CD34+ MNC counts. CONCLUSION: Circulating EPCs are significantly reduced in hemodialysis patients, which might be related to impaired neovascularization and cardiovascular disease in these patients.     

尼古丁对人外周血内皮干细胞数量和活性的影响

目的　观察尼古丁影响出生后的血管新生是否与其影响外周血内皮干细胞(EPC)的数量和活性有关。方法　采用密度梯度离心法从外周血获取单个核细胞,将其接种在人纤维连接蛋白包被培养板,培养7d后,收集贴壁细胞,加入不同浓度尼古丁(分别为10 - 12 ,10 - 10 ,10 - 8,10 - 6 及10 - 4mol·L- 1)培养一定的时间(12 ,18,2 4 ,32及4 8h)。激光共聚焦显微镜鉴定FITC UEA Ⅰ和DiI acLDL双染色阳性细胞被认为是正在分化的EPC ,流式细胞仪检测其表面标志进一步鉴定EPC ,倒置荧光显微镜下计数。然后分别采用MTT比色法、改良的Boyden小室、体外血管生成试剂盒和粘附能力检测实验,观察EPC的增殖能力、迁移能力、体外血管生成能力和粘附能力。结果　尼古丁在浓度为10 - 12 ～10 - 8mol·L- 1范围内显著增加外周血EPC数量,改善外周血EPC的粘附能力、迁移能力、增殖能力和体外血管生成能力,并且EPC数量随尼古丁浓度增加而增加,10 - 8mol·L- 1浓度尼古丁EPC影响最为显著(P <0 .0 1,n =6 )。另外,10 - 8mol·L- 1尼古丁呈时间依赖地增加外周血EPC数量,改善外周血EPC功能。而尼古丁在浓度>10 - 6 mol·L- 1却减少外周血EPC数量,损害外周血EPC功能。结论　尼古丁可增加EPC数量并改善其功能,然而高浓度尼古丁或许对EPC具有毒性     

Endothelial progenitor cell differentiation using cryopreserved, umbilical cord blood-derived mononuclear cells

AIM: To investigate the endothelial differentiation potentiality of umbilical cord blood (UCB), we induced the differentiation of endothelial progenitor cells (EPC) from cryopreserved UCB-derived mononuclear cells (MNC). METHODS: MNC from cryopreserved UCB and peripheral blood (PB) were cultured in M199 medium with endothelial cell growth supplements for 14 d. EPC were characterized by RT-PCR, flow cytometry, and immunocytochemistry analysis. The proliferation of differentiated EPC was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, and vascular endothelial growth factor (VEGF) concentration was measured using an ELISA kit. Characteristics of UCB-derived EPC were compared with those of PB-derived EPC. RESULTS: A number of round-shaped cells were loosely attached to the bottom after 24 h culture, and numerous spindleshaped cells began to appear from the round-shaped ones on d 7. Those cells expressed endothelial markers such as, Flt-1/VEGFR-1, ecNOS, VE-cadherin, von Willebrand factor, and secreted VEGF. The patterns of endothelial markers of EPC from PB and UCB did not show striking differences. The results of the proliferation and secretion of VEGF were also similar. CONCLUSION: We successfully cultured UCB cells stored at -196 Celsius degree into cells with the quality of endothelial cells. Those EPC could be used for angiogenic therapeutics by activating adjacent endothelial cells and enhancing angiogenesis.     

The effects of HMG-CoA reductase inhibitor on vascular progenitor cells

Circulating bone marrow-derived vascular progenitor cells contribute to angiogenesis, atherosclerosis, and the response to vascular injury. These vascular progenitor cells consist of two cell groups, endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs). Although HMG-CoA reductase inhibitors (statins) have been reported to inhibit atherosclerosis partially by increased EPCs, the effects of statins on SMPCs are unclear. Therefore, we investigated the relationship between EPCs and SMPCs and whether pravastatin has atheroprotective effects on SMPCs. Peripheral mononuclear cells (MNCs) were isolated and cultured on fibronectin-coated dishes in SMPC medium. MNCs were stained with acetylated low density lipoprotein and lectin, or alpha-smooth muscle actin, and cell numbers were counted. mRNA expression and vascular endothelial growth factor (VEGF) protein synthesis of MNCs were evaluated. Pravastatin significantly increased the number of EPC and decreased the number of SMPC. mRNA expression of VEGF, endothelial nitric oxide synthase, VEGF receptor-2 (KDR), and Akt were up-regulated, and VEGF secretion was increased by pravastatin. The present study demonstrated that pravastatin has promotive effects on the differentiation from MNCs to EPC cells, while inhibitory effects to SMPC cells. Our findings suggest a previously unreported mechanism of the effect of statin therapy on vascular progenitor cells.