丁苯酞对急性缺血性脑卒中患者外周血EPCs的影响

目的观察丁苯酞对急性缺血性脑卒中(AIS)患者外周血内皮祖细胞(EPCs)的影响,并分析其临床疗效。方法收集2014年4月至2015年4月我院神经内科收治的AIS患者88例,随机分为常规治疗组(对照组)和丁苯酞治疗组(观察组),每组44例。流式细胞仪检测外周血EPCs细胞,美国国立卫生研究院卒中量表(NIHSS)评估患者神经功能,比较两组的临床疗效以及不良反应发生率。结果两组治疗前外周血EPCs细胞计数比较差异无统计学意义(P>0.05),对照组治疗14 d后,EPCs细胞逐渐减低(P<0.05),而观察组治疗7 d后,外周血EPCs细胞逐渐升高(P<0.05),到14 d后达到高峰(P<0.05),治疗7、14、28 d后,观察组患者外周血EPCs细胞计数均高于对照组(P<0.05、P<0.01)。治疗前与治疗7 d后,两组NIHSS评分比较差异无统计学意义(P>0.05),治疗14 d后和28 d疗程结束后,观察组患者NIHSS评分均低于对照组(P<0.05、P<0.01)。观察组临床治疗有效率为93.2%,显著优于对照组的81.8%(P<0.05)。对照组临床治疗不良反应率为9.1%,观察组临床治疗不良反应率为11.4%,两组间比较差异无统计学意义(P>0.05)。结论丁苯酞可以动员AIS患者外周血EPCs细胞,有利于神经功能的恢复,临床疗效显著,使用安全,具有一定的临床应用价值。     

有氧运动对大鼠外周血EPCs G_1/S转换信号通路相关基因mRNA表达的影响

目的:研究有氧运动对EPCs细胞周期关键转换点G1/S转换信号通路相关基因mRNA表达的影响,从分子水平探讨有氧运动影响外周血EPCs增殖的机制。方法:两月龄雄性SD大鼠20只,按体重分层随机分为空白对照组和有氧运动组.。采用递增负荷跑台运动,每天训练1次,每周6天,由15 m/min(持续25 min)递增至26 m/min(持续50 min),跑台坡度为10%,共训练8周。运动方案结束后,采集大鼠外周血得到单个核细胞并诱导其分化获取EPCs,培养12天后收集贴壁EPCs进行全基因组检测,检测结果采用GeneSpring11.0芯片软件进行分析,对运动组与对照组相比差异表达基因进行信号通路分析。运动组/对照组基因差异表达倍数大于2倍视为表达具有差异,且表达上调;对照组/运动组基因差异表达倍数大于2倍视为表达具有差异,且表达下调。结果:在G1/S转换信号通路的10个相关基因中,Fbxo5 mRNA、Ccne1 mRNA、Orc1lmRNA、Dhfr mRNA、Pola1 mRNA、Pcna mRNA表达上调,Tyms mRNA、Cdc45l mRNA、Cdc6mRNA、Cdt1 mRNA无明显改变。结论:有氧运动可通过改变EPCs细胞周期G1/S转换相关基因Fbxo5、Ccne1、Orc1l、Dhfr、Pola1、Pcna mRNA表达,促使细胞增殖的关键点由DNA合成前期向DNA合成期转化,并为DNA的复制做充分准备,有利于EPCs细胞周期的缩短。     

Transplanted blood-derived endothelial progenitor cells (EPC) enhance bridging of sheep tibia critical size defects

The angiogenic events that accompany bone regeneration function as a “limiting factor” and are the primary regulatory mechanisms that direct the healing process. The general aim of this study was to test whether blood-derived progenitor cells that have endothelial characteristics (EPC), when applied to a large segmental defect, would promote bone regeneration. We established a critical-sized gap platform in sheep tibiae. Our model system takes advantage of the physiological wound healing process that occurs during the first two weeks following injury, and results in the gap being filled with scar tissue. EPC were expanded ex-vivo and 2 × 10⁷ cells/0.2 ml were implanted into a wedged-shaped canal excavated in the fibrotic scar tissue. Sham treated sheep served as controls. Bone regeneration was followed every two weeks for three months by X-ray radiography. At the end of the experimental period, the regenerating segments were subjected to micro-computed tomographic (μCT) analysis. While minimal bone formation was detected in sham-treated sheep, six out of seven autologous EPC-transplanted sheep showed initial mineralization already by 2 weeks and complete bridging by 8–12 weeks post EPC transplantation. Histology of gaps 12 weeks post sham treatment showed mostly fibrotic scar tissue. On the contrary, EPC transplantation led to formation of dense and massive woven bone all throughout the defect. The results of this preclinical study open new therapeutic opportunities for the treatment of large scale bone injuries.     

木瓜酶促进大鼠血管内皮细胞成血管的功能研究

目的　通过体外鼠细胞模型实验,检测木瓜酶在体外培养环境下对血管内皮细胞成血管向分化的促进功能。方法　采用P3代大鼠血管内皮细胞,将木瓜酶溶解于完全培养基内,按照10 μg/ml、50 μg/ml、100 μg/ml浓度配置含木瓜酶的完全培养基,与空白对照组共同培养0、7、14 d,通过逆转录聚合酶链反应（qRT-PCR）法检测成血管分化相关基因VEGF（血管内皮生长因子）、CD31（血小板-内皮细胞粘附分子）的表达水平,通过CCK-8法检测不同组细胞的增殖活力。统计学方法为方差分析。结果　经14 d培养,添加木瓜酶的细胞VEGF、CD31表达水平及增殖能力显著高于空白对照组,木瓜酶浓度达到50 μg/ml时VEGF表达水平达到最高,为空白对照组的3.28倍,CD31表达水平随着木瓜酶浓度升高而升高,最高达到空白对照组的3.66倍。结论　适宜浓度的木瓜酶能够在体外促进内皮细胞的成血管分化及增殖,表明木瓜酶对血管组织形成具有一定的促进作用。     

Tumour vascularization: sprouting angiogenesis and beyond

Tumour angiogenesis is a fast growing domain in tumour biology. Many growth factors and mechanisms have been unravelled. For almost 30 years, the sprouting of new vessels out of existing ones was considered as an exclusive way of tumour vascularisation. However, over the last years several additional mechanisms have been identified. With the discovery of the contribution of intussusceptive angiogenesis, recruitment of endothelial progenitor cells, vessel co-option, vasculogenic mimicry and lymphangiogenesis to tumour growth, anti-tumour targeting strategies will be more complex than initially thought. This review highlights these processes and intervention as a potential application in cancer therapy. It is concluded that future anti-vascular therapies might be most beneficial when based on multimodal anti-angiogenic, anti-vasculogenic mimicry and anti-lymphangiogenic strategies.     

Transplantation of VEGF165-gene-transfected endothelial progenitor cells

Background: To study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis. Methods: We constructed a recombinant adenoviral vector carrying the VEGF165 gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells (EPCs) were isolated from rat bone marrow by using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (Dil) before transplantation. An experimental rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups: A (n = 25), Ad-VEGF165/EPC-transplantation group, which received 1 ml (106) of Ad-VEGF165/EPCs; B (n = 25), EPC-transplantation group, which received 1 ml (106) of EPCs; C (n = 25), Ad/EPC-transplantation group, which received 1 ml (106) of Ad/EPCs; D (n = 25), control group, which received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction (PCR) was used to detect the expression level of VEGF mRNA; and western blotting was used to measure thrombosis and changes in VEGF protein expression. Hematoxylin-eosin (HE) staining and immunohistochemical staining were performed to detect recanalization, and neovascularization was observed by scanning electron microscopy. The capillary density was quantitatively determined by counting the capillaries under a high-power microscope. SPSS 11.5 software used for analysis, and differences at P < 0.05 were considered to be significant. Results: The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfection ratio that promoted the growth of EPCs. After transfection, the EPCs secreted the VEGF protein. After transplantation, the in vivo survival of EPCs and their differentiation into endothelial cells were determined by detecting the fluorescence associated with the Dil stain. VEGF mRNA was expressed in groups A, B, C, and D after transplantation, and the VEGF mRNA level in group A was significantly higher than those in groups B, C, and D (P < 0.05); the VEGF mRNA levels in groups B and C were significantly higher than those in group D (P < 0.05), and there was no statistical significance between the VEGF mRNA levels in groups B and group C. The recanalization capillary density in group A was significantly higher than those in groups B, C (P < 0.05), and D (P < 0.01); the recanalization capillary densities in groups B and C were significantly higher than that in group D (P < 0.05). Moreover, there was no statistical significant difference between the values for groups B and C. Neovascularization was detected by immunohistochemical staining using the antibody for vWF, which is a component of endothelial cells. Conclusion: The EPCs were successfully transfected by Ad-VEGF165. A suitable transfection ratio can improve the efficiency of EPCs, improving the possibility of promotion of angiogenesis after transplantation. Transfected EPCs caused accelerated organization and recanalization of vein thrombi.     

人脂肪组织来源内皮祖细胞的分离、培养及鉴定方法的实验研究

目的探讨人脂肪组织来源的内皮祖细胞的分离、培养及鉴定的方法。方法脂肪抽吸术获取人体脂肪组织,消化法得到的细胞接种在Fibronectin包被的培养皿内,用含2%胎牛血清的DMEM培养,传至第2代,分诱导组(EGM-2,2?S,VEGF,bFGF等)和非诱导组(DMEM,2?S)两组进行培养。免疫细胞荧光分别检测两组的CD34、vWF和PECAM-1表达;流式细胞仪分别检测两组的CD34、CD45、CD133和PECAM-1表达率;荧光显微镜观察细胞摄取DiI-ac-LDL的功能;诱导组细胞接种于甲基纤维素半固体培养基进行三维培养,观察血管样结构形成情况。结果诱导组细胞12d后呈现内皮细胞典型的铺路石样形态,未诱导组细胞未观察到这种变化;免疫细胞荧光显示诱导组vWF、PECAM-1表达阳性,未诱导组细胞CD34表达阳性;流式细胞仪检测显示诱导组PECAM-1阳性率为(67.41±13.35)%,明显高于非诱导组的(6.73±2.21)%(P<0.01),非诱导组CD34阳性率为(72.39±13.45)%,明显高于诱导组的(16.06±3.86)%(P<0.01);荧光显微镜观察显示诱导组细胞具有摄取DiI-ac-LDL的功能;诱导组细胞三维培养形成"树枝样"分叉结构。结论建立了一种从人脂肪组织分离、培养EPCs的方法,并对其表型进行鉴定,有望为血管组织工程提供新的种子细胞来源。     

Bim在内皮祖细胞自然凋亡中的作用

目的研究前凋亡因子Bim在内皮祖细胞（EPCs）自然凋亡中的作用。方法采集健康产妇产后2h脐静脉血,6%羟乙基淀粉沉淀及密度梯度离心法提取脐血中单个核细胞,培养10d,免疫荧光法及免疫组化法鉴定EPCs。采用流式细胞仪检测EPCs凋亡率,RT-PCR分析不同时间点（0,6,12,24,48h）BimmRNA的表达。结果体外培养的EPCs随培养时间发生自然凋亡,各时间点细胞凋亡百分比较对照组（0h）高;RT-PCR显示在一定时间内前凋亡因子BimmRNA随培养时间延长表达增高。结论 Bim参与了EPCs自然凋亡的过程,是重要的促细胞凋亡因子。     

细胞骨架F-actin在层流剪切应力诱导EPCs内皮分化中的作用

目的探讨细胞骨架F-actin在层流剪切应力促内皮祖细胞(endothelial progenitor cells,EPCs)向内皮分化中的作用。方法对大鼠骨髓来源的EPCs施以层流剪切应力(1.2 Pa),以荧光定量RT-PCR及流式细胞术检测特异性内皮细胞标记分子vWF、CD31 mRNA及蛋白的表达来反映EPCs分化程度;以免疫荧光染色观测F-actin的排列情况;应用Ras GTPase Pull-Down方法检测Ras活性。结果层流剪切力处理后,EPCs分化标记vWF及CD31的基因及蛋白表达较静止组明显升高(P<0.05),细胞骨架F-actin发生重排,Ras活性明显增高。细胞骨架稳定剂Jasplakinolide(JAS)及细胞骨架松弛剂Cytochalasin D(CytoD)预处理均不同程度地抑制了层流剪切应力所致的细胞骨架的重排、Ras活性的上调及EPCs分化效应(P<0.05),而过表达Ras则对层流剪切应力诱导的EPCs分化有明显促进作用(P<0.05)。结论一定大小的层流剪切应力可促进EPCs向内皮细胞分化,其机制可能与层流剪切应力重塑细胞骨架F-actin,进而影响Ras活性有关;这对于揭示受损血管内皮修复的具体机制、阐明动脉粥样硬化等心血管疾病的发病机理及临床防治此类疾病具有一定的意义。     

Apelin and vascular endothelial growth factor are associated with mobilization of endothelial progenitor cells after acute myocardial infarction

This study was designed to determine the levels of early endothelial progenitor cells(EPCs),apelin,vascu-lar endothelial growth factor(VEGF) and stromal cell-derived growth factor-1(SDF-1) after acute myocardial infarction(AMI),and to investigate the relationships between these cytokines and early EPCs.Early EPCs,de-fined as CD133+,KDR+,and CD34+ cells,were quantified by flow cytometry.The levels of early EPCs and those cytokines in AMI patients were significantly different from those with coronary artery disease or controls(P < 0.05).Plasma apelin levels were inversely correlated with Gensini score and early EPCs(both P < 0.01).Early EPCs,VEGF and SDF-1 showed different patterns of changes in AMI patients during the first 24 h.The trend in the change of early EPCs was proportionally correlated with that of VEGF(P < 0.05).AMI patients exhibited in-creased early EPCs with remarkably decreased apelin levels and enhanced VEGF levels.