疏肝活血方含药血清对内皮祖细胞数量及功能的影响

目的：观察疏肝活血方含药血清对大鼠骨髓来源的血管内皮祖细胞（EPCs）数量及功能的影响。方法：从大鼠骨髓中获取单个核细胞,体外培养9d后,收集贴壁细胞,加入低、中、高浓度的疏肝活血方及其拆方的含药血清,培养12、24、48h。激光共聚焦显微镜鉴定,FITC-UEA-I和Dil-acLDL双染色阳性细胞为正在分化的EPCs,并在倒置荧光显微镜下计数,分别采用黏附能力测定、MTT比色法及改良的Boyden小室实验来观察疏肝活血中药对EPCs黏附能力、增殖能力和迁移能力的影响,用流式细胞仪检测PI-/AnnexinⅤ＋的凋亡细胞。结果：疏肝活血方能明显促进EPCs的分化,提高EPCs的数量,明显改善EPCs的黏附、增殖及迁移能力,减少EPCs的凋亡率,中浓度组培养24h达到作用的高峰（P〈0.01）。结论：疏肝活血方能促进EPCs数量的增加、功能的改善,减少EPCs的凋亡。     

环孢素A对抑制外周血内皮祖细胞增殖活性的作用

目的 观察环孢素A(CsA)对培养的人外周血内皮祖细胞(EPCs)增殖和凋亡的影响.方法 采用密度梯度离心法分离外周血单个核细胞,贴壁培养获得EPCs.比色法测定EPCs增殖能力;TUNEL染色检测EPCs凋亡情况;逆转录聚合酶链法检测EPCs凋亡相关基因的表达.观察不同浓度的CsA对EPCs增殖能力的作用;用10 mg/L的CsA干预24 h和48 h对EPCS增殖能力的影响,以及干预48 h对EPCs凋亡以及凋亡蛋酶-3(easpase-3)、凋亡调控基因bcl2和bax表达的影响.结果 100μg/L的CsA能明显抑制EPCs的增殖,CsA在10～10000μg/L浓度范围内,抑制作用成浓度依赖性.10 mg/L的CsA明显增加EPCs的凋亡和caspase-3基因表达,抑制bcl2基因表达而减小bcl2/bax比值.结论 CsA能明显抑制培养的人外周血EPCs增殖、增加其凋亡;抑制bcl2基因表达是其诱导EPCs凋亡的机制之一.     

长期血液透析患者内皮祖细胞的变化及其影响因素分析

目的 检测长期面液透析患者外周血内皮祖细胞数量(EPCs)和功能的改变,并探讨其与微炎症、贫血、高同型半胱氨酸血症各影响因素的关系.方法 采用Ficoll密度梯度离心法分离培养慢性肾功能衰竭长期血液透析患者组和健康组的外周血单个核细胞,将其接种在人纤维连接蛋白包被培养板,7天后取贴壁细胞进行Eli-LDL和FITC-UEA-I双染色,并通过流式细胞仪检测其表面标志CD34、CD133、KDR,以鉴定EPCs.采用改良Boyden小室、黏附功能检测评价其迁移和黏附能力,并与患者血红蛋白(Hb)、血清半胱氨酸(Hey)、C反应蛋白(CRP)含量进行多元线性逐步回归分析.结果 长期血液透析患者EPCs 的数量和迁移、黏附功能都低于正常健康者,差异有统计学意义(P<0.05);患者血清CRP、Hcy含量高于正常对照组(P<0.05),与患者EPcs的数量和迁移、黏附功能分别呈负相关(P<0.05);患者Hb含量低于正常对照组(P<0.05),与患者EPCs的数量和黏附功能分别呈正相关;多元线性逐步回归分析发现血清CRP和Hcy是患者EPCs的数量和功能的独立影响因素.结论 长期血液透析患者的EPCs数量和功能降低,存在血管新生和内皮修复的缺陷,增加了患冠心病的风险,其中血清CRP和Hey是患者EPCs的数量和功能的独立影响因素.     

人VEGF165基因转染人外周血内皮祖细胞的实验研究

目的 探讨人血管内皮细胞生长因子165(hVEGF165)基因转染对人外周血内皮祖细胞(EPCs)的影响.方法 体外分离、培养、鉴定人外周血EPCs.实验分脂质体介导pcDNA3.1-hVEGF165质粒转染组,pcDNA3.1空质粒转染组,窄白对照组.ELISA法和硝酸还原酶法分别测定各组上清液中VEGF和一氧化氮(NO)的含量;噻唑蓝(MTT)检测它们对EPCs增殖的影响.结果 FITC-UEA-I和DiI-ac-LDL双染色阳性细胞为正在分化的EPCs,脂质体介导pcDNA3.1-hVEGF165质粒转染组EPCs培养基上清液中VEGF和NO表达量明显高于其他两组(P＜0.01);VEGF质粒转染对EPCs的增殖无明显影响.结论 人外周血EPCs可以成功转染hVEGF165基因.同时能表达一定浓度的VEGF蛋白,并可促进NO的分泌,而对EPCs的活性无明显影响.该研究为进一步研究VEGF165基因和EPCs结合治疗缺血性疾病提供了实验依据.     

酸性成纤维细胞生长因子对人脐血内皮祖细胞的影响

目的 观察酸性成纤维细胞生长因子(aFGF)对体外培养内皮祖细胞(EPCs)数量、功能及凋亡的影响.方法 密度梯度离心法获取人脐血单个核细胞(MNCs),培养6 d后,收集贴壁细胞流式细胞仪和激光共聚焦显微镜鉴定EPCs.并向贴壁细胞分别加入aFGF 2、5、10μg/L干预培养24 h,同时对作用效果最为显著的组(aFGF 5μg/L组)分别培养6、12、24、48 h,分别观察EPCs数量、增生、迁移、黏膜及凋亡状况,从而对其时效关系进行观察.结果 与对照组比较,不同浓度的aFGF可以显著提高EPCs的数量、生物学功能并抑制其凋亡(P<0.05);本研究5μg/L aFGF作用24 h时对EPCs数量、生物学功能及凋亡抑制的影响最为显著(P<0.05).结论 aFGF增加体外培养条件下EPCs的数目、改善其生物学功能并抑制其凋亡.     

miR-126对内皮祖细胞增殖和迁移的影响以及靶基因的检测

目的 研究miR-126(micro RNA-126)对大鼠骨髓源性内皮祖细胞(endothelial progenitor cells,EPCs)增殖和迁移能力的影响,并探讨miR-126新的靶基因.方法 采用电转的方法,在EPCs中转染对照寡核苷酸和miR-126的模拟物或抑制物.噻唑蓝(MTT)法检测细胞增殖,划痕和transwell实验检测细胞迁移能力的改变.microRNA靶基因预测软件TargentScan在线分析miR-126的靶基因,并进一步用Western blot检测靶基因的表达变化.结果 (1) miR-126模拟物在转染后24 h对EPCs的增殖有促进作用(P＜0.01),转染后48和72 h,miR-126对EPCs增殖没有影响.(2)划痕和transwell实验证实miR-126模拟物可以促进EPCs的迁移(P＜0.01),miR-126抑制物抑制EPCs的迁移(P＜0.01).(3)TargetScan在线软件预测KANK2是miR-126的靶基因.(4) Western blot检测结果显示miR-126模拟物抑制KANK2的表达,miR-126抑制物促进KANK2的表达.结论 miR-126对EPCs的增殖有一过性的促进作用,miR-126可以促进EPCs的迁移能力并靶向KANK2蛋白,抑制KANK2蛋白的表达.     

ADSCs培养上清液对成纤维细胞的生物学影响

目的:分离培养脂肪干细胞(adipose-derived stem cells,ADSCs),探讨其培养上清液对成纤维细胞的生物学作用。方法:从人腹部脂肪组织中分离、培养ADSCs,收集其上清液培养人成纤维细胞。MTT法测定成纤维细胞的增殖;AnexinⅤ/PI双染色法测定成纤维细胞的凋亡;体外细胞划痕法测定其迁移能力。结果:培养获得ADSCs,ADSCs培养上清液实验组成纤维细胞的增殖及迁移均明显大于对照组(P<0.05或P<0.01);同时其凋亡率也低于对照组(P<0.05)。结论:ADSCs培养上清液能促进成纤维细胞的增殖、迁移及抑制凋亡发生。     

糖基化终末产物对人外周血内皮祖细胞生物学特性的影响

Objective In addition to be involved in the angiogenesis, endothelial progenitor cells (EPCs) have roles in endothelium repairing, wound healing, and for protecting blood vessels from restenosis, Advanced glycation end products (AGEs) facilitate the development and progression of atherosclerosis, diabetes associated vascular complications and uremia through various mechanisms such as damaging the endothelium, promoting leukocyte adhension, increasing the aggregation of platelets, and stimulating the proliferation of vascular smooth muscles. This study was designed to explore whether AGEs have effects on biological characteristics of EPCs in cultured human peripheral blood cells. Methods Total mononuclear cells (MNCs), isolated from human peripheral blood by density gradient centrifugatian and adherence cells filtration, were incuba-ted in fibronectin-coated culture dishes. Endothelial cells were identified by means of the adsorption of ulex eurepaeus-aggluti-nin- Ⅰ (UEA- Ⅰ) labelled with fluorescein isothiacyanate (FITC) and Dil-acLDL internalization. Four days later,various con-centrations of AGEs were added to the adherent cells and remained for48 hours. MTT assay and Boyden chamber were used for observing the proliferation and migration of EPCs. Human fibronectin was used to examine the adhesion ability of EPCs. Apop-tosis was induced in the EPCs with formaldehyde and Dnase Ⅰ as a positive control group. Annexin V-FITC/PI and TUNEL method of flow cytometry were used for evaluating the effects of AGEs on the rate of apeptosis in the EPCs. Results AGEs at high concentration decreased the number of EPCs independently (P < 0.01) ; reduced the proliferation (P < 0.01), migration (P<0.001) and adhesive capacity (P<0.05) of EPCs significantly,as well as increasing the apoptasis rate of EPCs in the early stage (P < 0.001). Conclusion AGEs may have adverse effects on EPCs from cultured human peripheral MNCs, such as decreasing their numbers and impairing their functions.     

表达人端粒酶逆转录酶小干扰RNA的增殖腺病毒对肝癌细胞的抑制作用

目的 观察表达人端粒酶逆转录酶小干扰RNA(hTERT-siRNA)的增殖腺病毒(ZD-hTERT)对人肝癌Bel-7402细胞增殖及凋亡影响.方法 ZD-hTERT、增殖腺病毒ZD-EGFP、表达hTERT-siRNA的增殖缺陷腺病毒Ad-hTERT、增殖缺陷腺病毒Ad-EGFP分别感染人肝癌Bel-7402细胞.Western blot法检测ElA表达;逆转录-聚合酶链反应(RT-PCR)、Western blot法检测hTERT表达;噻唑蓝(MTT)比色法检测细胞存活;结晶紫染色法检测细胞毒作用;原位末端标记法(TUNEL)检测凋亡.结果 感染ZD-hTERT、ZD-EGFP的Bel-7402细胞表达ElA;抑制hTERT表达作用依次为ZD-hTERT>Ad-hTERT>ZD-EGFP>Ad-EGFP;抑制Bel-7402细胞生长及细胞毒作用依次为ZD-hTERT>ZD-EGFP=Ad-hTERT>Ad-EGFP.感染ZD-hTERT、ZD-EGFP、Ad-hTERT、Ad-EGFP的Bel-7402细胞凋亡率(%)分别为(88.1±2.2)、(39.2±2.1)、(42.1±5.1)、(7.5±2.1),ZD-hTERT诱导凋亡作用最高(P<0.01).结论 表达hTERT-siRNA的增殖腺病毒能显著抑制人肝癌Bel-7402细胞hTERT基因表达,进而抑制其增殖,促进其凋亡.     

低氧诱导因子-1α激动剂对内皮祖细胞生物学行为的影响

目的 观察低氧诱导因子-1α( HIF-1α)的激动剂对兔骨髓内皮祖细胞(EPCs)的增殖和功能的生物学影响.方法 以不同浓度的HIF-1α的激动剂二甲基乙二酰基甘氨酸(DMOG)干预EPCs,检测其增殖活性、集落形成能力、一氧化氮(NO)的产量、迁移能力、衰老状态、成血管功能及相关蛋白的表达.结果DMOG能显著激活EPCs中HIF-1α蛋白表达,增加细胞的活性,且成剂量依赖关系,其中浓度500 μmol/L时增殖作用达到峰值.同时DMOG增加EPCs集落形成能力(P＜0.01),抑制细胞的衰老(P＜0.05),并显著提高EPCs的的迁移能力和体外成血管功能.结论 HIF-1α的激活剂能显著提高内皮祖细胞增殖能力、集落形成能力、迁移能力和成血管功能.     

Changes of the peripheral blood endothelial progenitor cells in peritoneal dialysis patients and its impact factors

Objective To investigate the number and activities changes of peripheral blood endothelial progenitor cells (EPCs) in continuous ambulatory peritoneal dialysis (CAPD) patients, and explore the connection between EPCs' number and the levels of advanced glycosylation end products (AGEs), homocysteine (Hcy) and C-reactive protein (CRP). Methods Twenty-five CAPD patients and thirty healthy volunteers were involved. Total mononuclear cells (MNCs) were isolated from peripheral blood of patients. EPCs were characterized as adherent cells by double staining of FITC-UEA-1 and DiL-AcLDL binding, and were further demonstrated by positive cells of CD34, CD133 and KDR using flow cytometry. The abilities of cell proliferation, adhesion and migration were further observed by fluorescent microscope. The correlations between the CEPCs' number and the levels of AGEs, Hcy andCRP were analyzed. Results The number and activities including migration and adhesion of EPCs in CAPD group were significantly lower than control group (P＜0.05). The levels of serum AGEs, Hcy and CRP in CAPD patients were increased (all P＜0.05) and had negative correlation with EPCs' number. Conclusions The number and activities of EPCs decrease in patients with CAPD, and EPCs' number is negatively correlated to the levels ofAGEs, Hcy and CRP.     

人脐血内皮祖细胞治疗裸鼠心肌梗死

目的 探讨人脐血内皮祖细胞(EPCs)移植治疗裸鼠心肌梗死的可行性.方法 采用淋巴细胞分离液提取人脐血单个核细胞(MNCs),应用添加诱导因子的培养基于体外诱导分化并于培养7 d后进行鉴定.采用20只裸鼠建立心肌梗死模型后,将体外诱导分化7 d并摄取CM-Dil的内皮祖细胞通过尾静脉注射进行细胞移植到实验组,对照组注射培养基.2周后计数心梗区域新生毛细m管密度及心梗面积并于荧光显微镜下观察新生血管的荧光.结果 体外诱导7 d后贴壁细胞CD34阳性率达(50.48±5.17)%,CDl33阳性率达(19.12±4.37)%.实验组平均梗死面积为(8.27±1.64)%,对照组为(14.30±2.84)%(t=-4.78,P<0.05);实验组每高倍视野平均新生血管密度为14.29±1.38,对照组为10.17±1.72(t=4.71,P<0.01);行荧光显微镜下观察实验组新生血管有红色荧光.讨论人脐血单个核细胞在体外诱导分化为内皮祖细胞,进行细胞移植到建立心梗模型的裸鼠后可在心梗区域形成新生血管,从而并改善梗死部位心脏功能.     

骨髓单个核细胞和骨髓源内皮祖细胞移植促进血流重建的比较研究

目的 比较骨髓单个核细胞(MNCs)和骨髓源内皮祖细胞(EPCs)移植促进血流重建的效果,探讨非内皮祖细胞在血流重建中的作用.方法 获取Lewis大鼠骨髓MNCs,部分MNCs在体外诱导分化为EPCs.采用Lewis大鼠建立单侧后肢缺血模型.建模后3 d,将模型鼠随机分为3组:(1)对照组(n=6),将0.8 mL D-Hank's液注入对照组大鼠缺血侧后肢;(2)MNC组(n=6),将8×10~6个骨髓MNC植入MNC组大鼠缺血侧后肢;(3)EPC组(n=6),将体外培养的8 × 10~6个EPC植入EPC组大鼠缺血侧后肢.细胞移植后3周行大鼠后肢动脉造影,检测缺血侧后肢侧支血管数;切取缺血侧后肢腓肠肌,分别行CD31和α-SMA免疫组化染色,计算毛细血管密度和小动脉密度.结果 MNC组毛细血管密度与EPC组差异无统计学意义[(31.67 ± 7.87)个/HP vs.(32.83±5.38)个/HP,P＞0.05].而均高于对照组(19.67 ± 4.80个/HP)(P＜0.05);MNC组侧支血管数与EPC组差异无统计学意义[(4.17±0.75)个vs.(4.50 4±1.38)个,P＞0.05],但均高于对照组[(2.50 ± 1.5)个](P＜0.05);MNC组小动脉密度与EPC组差异无统计学意义[(4.83 ± 1.47)个vs.(5.50 ± 2.35)个,P＞0.05],亦均高于对照组[(2.17±0.98)个](P＜0.05).结论 在骨髓干细胞移植治疗肢体缺血性疾病中,非内皮祖细胞在血流重建中所起的作用与EPC相似.     

Differentiation and proliferation of endothelial progenitor cells from canine peripheral blood mononuclear cells

BACKGROUND: The isolation, differentiation, and expansion of endothelial progenitor cells (EPCs) from peripheral blood have potential applicability in areas of therapeutic neovascularization, vascular repair, and tissue engineering. The purpose of the current study was to elucidate a simple method of isolation and differentiation of EPCs by defining the endothelial morphology, surface marker expression, and proliferative capacity of EPC outgrowth from canine peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: PBMCs were isolated from fresh canine blood and cultured in fibronectin-coated plates in which EPCs were identified from cell morphology and outgrowth characteristics. Cell surface markers were determined with flow cytometry analysis to identify differentiation of cultured and subcultured colonies. A hematologic counter with phase contrast microscopy was used to study cell growth curves of EPCs as compared with mature human coronary artery endothelial cells. RESULTS: During the first week of canine PBMC culture, cells were morphologically round and varied in size, but in the course of the second and third week of culture, the cells, respectively, became spindle-shaped and displayed an endothelium-like cobblestone morphology with outgrowth. CD34 was significantly decreased at 21 days as compared with 7 days culture (36.04% to 21.37%), whereas vWF (from 77.26% to 96.37%) and eNOS (from 0% to 14.97%) were significantly increased. VEGFR-2 was slightly increased, and P1H12 (CD146) was unchanged. Subcultured canine EPCs displayed a higher proliferation rate as compared to mature human coronary artery endothelial cells in the same culture conditions. CONCLUSIONS: These data demonstrate that canine EPCs can be isolated and cultured from the canine PBMC fraction. These outgrowth cells displayed characteristics of endothelial morphology with endothelial cell-specific surface markers. Furthermore, it was revealed that canine EPCs have a greater growth potential as compared to mature endothelial cells. This study suggests that PBMCs could be used as a source of EPCs for potential applications in tissue engineering and vascular therapy.     

Transplanted endothelial progenitor cells augment the survival areas of rat dorsal flaps

Endothelial progenitor cells (EPCs) have been identified in peripheral blood, and have been reported to be incorporated into ischemic regions such as the ischemic hindlimb. In this study, we examined whether or not transplantation of EPCs is useful for salvaging surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted EPCs and that of mature endothelial cells (ECs). ECs obtained from the aorta of rats by explantation and passaged several times were used in the present study. EPCs were obtained from the blood of rat hearts. The blood samples were separated by density gradient centrifugation. Light-density mononuclear cells (MNCs) were collected and cultured on plastic plates coated with rat plasma vitronectin. Cells attached at day 7 of culture were deemed to be EPCs. Then PBS (control), ECs, or EPCs (3.0 x 10(5) suspended in 1.0 ml PBS) were injected at the middle of a flap. Seven days after surgery, the survival lengths of the flaps were evaluated. EPC-transplanted groups revealed statistically significant augmentation of survival length compared with the other two groups (p < 0.003). EPC-transplanted groups had significantly more angiographically detectable blood vessels (p < 0.003) and significantly higher capillary density (p < 0.03) than the other two groups. Confocal microscopy revealed that EPCs were incorporated into enhanced neovascularization. These results suggest that transplantation of EPCs may be useful for salvaging surgical flaps, and EPCs are superior to ECs in neovascularization ability.