缺氧态下Galectin-3对外周血内皮祖细胞源性血管内皮细胞的影响及机制研究
发布时间：2018-08-20 16:51
【摘要】：背景：血管发生曾被认为只存在于胚胎期原始血管网形成过程中。后证实出生后的新血管形成并不仅限于血管新生,也包括胚胎期的血管发生机制。传统观点认为,新生血管形成主要包括两种机制,血管新生与血管发生：血管新生(angiogenesis)是指通过原存在血管的成熟内皮细胞的增殖和迁移,以出芽方式长出新的毛细血管的过程,这一过程曾被认为是出生后血管形成的唯一机制,而血管发生(vasculogenesis)则是通过骨髓起源的内皮祖细胞(EPCs)聚集到缺血损伤部位并分化为血管内皮细胞(endothelialeens, ECs),并组织成血管的过程。在临床应用中,干细胞(血管中EPCs)移植新生血管为治疗缺血性疾病提供了另一个选择。 研究目的：将人外周血内皮祖细胞分离、培养并定向诱导分化为内皮细胞,研究其体外诱导培养的条件及生物学活性,探索细胞生长最佳干预期,为人外周血EPCs的研究奠定基础。同时,在缺氧态下观察Galectin-3对内皮祖细胞源性血管内皮细胞增殖能力的影响,研究干细胞移植治疗缺血性疾病的相关机制。 方法：本实验研究主要分为三个方面：一、人外周血内皮祖细胞的分离、培养、鉴定及活性研究：无菌条件下采集健康捐献者外周静脉血20ml,采用密度梯度离心法(Ficoll法)分离人外周血单个核细胞,通过细胞计数,台盼蓝染色鉴定细胞活力后,将其接种在预先包被有人纤维连接蛋白的培养皿中培养。每日于倒置相差显微镜下观察细胞的生长形态学变化,观测细胞生长情况,进行EPCs特征的鉴定：VEGFR-2、CD133、vWF的免疫荧光检测；行细胞功能实验：摄取acLDL和结合UEA-1检测。 二、缺氧态下内皮祖细胞的生长观察：收集培养7天的内皮祖细胞,随机分配为氯化钴(CoCl2)浓度分别为0,50,75,100,150,200,300μmol/L的浓度组,分别培养6h,12h,24h,48h,72h,观察在不同时间不同缺氧浓度下内皮祖细胞的生长情况,MTT法观察其生长情况,确定缺氧环境的最佳干预期及浓度。 三、缺氧态下Galectin-3对内皮细胞增殖能力的影响：收集贴壁的成熟血管内皮细胞细胞,在100umol/L氯化钴浓度下,随机分为Galectin-3终浓度各浓度组(共6组)：在培养液中加入终浓度为0u g/ml,1μg/ml,2.5μg/ml,5μ g/ml,10μg/ml,15μg/ml Galectin-3培养24小时,MTT比色法观察缺氧态下不同浓度Galectin-3对内皮细胞增殖能力的影响。 结果：分离获得的单个核细胞,细胞计数、台盼蓝染色后,活细胞百分率在(96±3)%,培养4天后绝大多数细胞贴壁生长,多为圆形或椭圆形；第5天起,部分较大圆形细胞转化为梭状内皮样细胞；第7天可见部分细胞聚集成集落；10天左右,细胞变为条索状,相互交联。在培养的第7天,细胞生长增殖情况最佳。对细胞性功能和特征测定,细胞的内皮祖细胞表面特异性标志因子VEGFR-2、CD133与vWF表达情况尚佳,摄取acLDL和结合UEA-1检测实验结果显示强阳性。细胞继续培养约1月时可见细胞成典型铺路石样改变。随着细胞培养液中CoCl2浓度的增加及培养时间的延长,细胞生长抑制率呈增加趋势,其中CoCl2100μmol/L,培养24h细胞的生长情况较其他组好。Galectin-3在缺氧态下亦可明显增加血管内皮细胞增殖能力,呈一定的量效关系,其中5.0μg/ml和10μg/ml浓度的可明显促进内皮细胞的增殖能力(P0.05)。 结论：1、采用Ficoll法从人外周血中可以成功分离出具有增殖分化潜能的内皮祖细胞,活细胞百分率在(96±3)%。2、在缺氧态下(CoCl2100μmol/L),细胞培养24h的生长情况最佳。3、在同一Galectin-3浓度下,缺氧态较常氧态对细胞增殖的促进作用更强。
[Abstract]:BACKGROUND: Angiogenesis was once thought to exist only in the process of embryonic primitive angiogenesis. It has been proved that postnatal angiogenesis is not limited to angiogenesis, but also includes the mechanism of embryonic angiogenesis. Genesis is the process by which new capillaries are sprouted by the proliferation and migration of mature endothelial cells that originate from blood vessels. This process was once thought to be the only mechanism of post-natal angiogenesis, whereas vasculogenesis is the accumulation of bone marrow-derived endothelial progenitor cells (EPCs) into ischemic sites. Endothelial cells (ECs) differentiate into vascular endothelial cells (ECs) and organize the process of angiogenesis. In clinical applications, stem cells (EPCs) transplantation of new blood vessels provides another option for the treatment of ischemic diseases.
OBJECTIVE: To isolate, culture and differentiate human peripheral blood EPCs into endothelial cells (EPCs), to study the conditions and biological activities of EPCs in vitro, and to explore the optimal intervening period of EPCs in human peripheral blood. The effect of stem cell transplantation on ischemic disease is studied.
Methods: This study was divided into three aspects: 1. Isolation, culture, identification and activity of human peripheral blood endothelial progenitor cells (EPCs): 20 ml peripheral venous blood from healthy donors was collected under aseptic conditions, and human peripheral blood mononuclear cells were isolated by density gradient centrifugation (Ficoll method). The cells were identified by cell count and trypan blue staining. After viability, the cells were cultured in a dish pre-coated with human fibronectin. The morphological changes of the cells were observed daily under an inverted phase contrast microscope, and the growth of the cells was observed. Test.
2. Growth observation of EPCs under hypoxia condition: EPCs were collected and cultured for 7 days and randomly assigned to the concentration groups of 0,50,75,100,150,200,300 micromol/L of cobalt chloride (CoCl2). The EPCs were cultured for 6 h, 12 h, 24 h, 48 h and 72 h respectively. The growth of EPCs was observed by MTT method. The best dry anticipation and concentration of anoxic environment were determined.
3. Effects of Galectin-3 on the proliferation of endothelial cells under hypoxia: Adherent mature vascular endothelial cells were collected and randomly divided into six groups at 100 umol/L of cobalt chloride concentration. The final concentration of Galectin-3 was 0 u g/ml, 1 ug/ml, 2.5 ug/ml, 5 ug/ml, 10 ug/ml, 15 ug/ml of Galectin-3 was added into the culture medium. 24 hours, MTT colorimetric method was used to observe the effect of different concentrations of Galectin-3 on endothelial cell proliferation under hypoxia.
Results: Mononuclear cells were isolated and counted. After trypan blue staining, the percentage of living cells was (96 6550 Cells grew and proliferated best on the 7th day of culture. The expression of endothelial progenitor cell surface-specific markers, such as VEGFR-2, CD133 and vWF, was still good. The results of acLDL uptake and UEA-1 binding assay showed that the cells were strongly positive. With the increase of the concentration of CoCl2 in the culture medium and the prolongation of the culture time, the growth inhibition rate of the cells increased. The growth of the cells cultured for 24 hours was better than that of the other groups. Galectin-3 could also significantly increase the proliferation ability of vascular endothelial cells under hypoxia. The 5 g/ml and 10 g/ml concentrations significantly promoted the proliferation of endothelial cells (P0.05).
CONCLUSION: 1. Endothelial progenitor cells with proliferative and differentiative potential can be successfully isolated from human peripheral blood by Ficoll method. The percentage of living cells is (96 65
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329
本文编号：2194328
[Abstract]:BACKGROUND: Angiogenesis was once thought to exist only in the process of embryonic primitive angiogenesis. It has been proved that postnatal angiogenesis is not limited to angiogenesis, but also includes the mechanism of embryonic angiogenesis. Genesis is the process by which new capillaries are sprouted by the proliferation and migration of mature endothelial cells that originate from blood vessels. This process was once thought to be the only mechanism of post-natal angiogenesis, whereas vasculogenesis is the accumulation of bone marrow-derived endothelial progenitor cells (EPCs) into ischemic sites. Endothelial cells (ECs) differentiate into vascular endothelial cells (ECs) and organize the process of angiogenesis. In clinical applications, stem cells (EPCs) transplantation of new blood vessels provides another option for the treatment of ischemic diseases.
OBJECTIVE: To isolate, culture and differentiate human peripheral blood EPCs into endothelial cells (EPCs), to study the conditions and biological activities of EPCs in vitro, and to explore the optimal intervening period of EPCs in human peripheral blood. The effect of stem cell transplantation on ischemic disease is studied.
Methods: This study was divided into three aspects: 1. Isolation, culture, identification and activity of human peripheral blood endothelial progenitor cells (EPCs): 20 ml peripheral venous blood from healthy donors was collected under aseptic conditions, and human peripheral blood mononuclear cells were isolated by density gradient centrifugation (Ficoll method). The cells were identified by cell count and trypan blue staining. After viability, the cells were cultured in a dish pre-coated with human fibronectin. The morphological changes of the cells were observed daily under an inverted phase contrast microscope, and the growth of the cells was observed. Test.
2. Growth observation of EPCs under hypoxia condition: EPCs were collected and cultured for 7 days and randomly assigned to the concentration groups of 0,50,75,100,150,200,300 micromol/L of cobalt chloride (CoCl2). The EPCs were cultured for 6 h, 12 h, 24 h, 48 h and 72 h respectively. The growth of EPCs was observed by MTT method. The best dry anticipation and concentration of anoxic environment were determined.
3. Effects of Galectin-3 on the proliferation of endothelial cells under hypoxia: Adherent mature vascular endothelial cells were collected and randomly divided into six groups at 100 umol/L of cobalt chloride concentration. The final concentration of Galectin-3 was 0 u g/ml, 1 ug/ml, 2.5 ug/ml, 5 ug/ml, 10 ug/ml, 15 ug/ml of Galectin-3 was added into the culture medium. 24 hours, MTT colorimetric method was used to observe the effect of different concentrations of Galectin-3 on endothelial cell proliferation under hypoxia.
Results: Mononuclear cells were isolated and counted. After trypan blue staining, the percentage of living cells was (96 6550 Cells grew and proliferated best on the 7th day of culture. The expression of endothelial progenitor cell surface-specific markers, such as VEGFR-2, CD133 and vWF, was still good. The results of acLDL uptake and UEA-1 binding assay showed that the cells were strongly positive. With the increase of the concentration of CoCl2 in the culture medium and the prolongation of the culture time, the growth inhibition rate of the cells increased. The growth of the cells cultured for 24 hours was better than that of the other groups. Galectin-3 could also significantly increase the proliferation ability of vascular endothelial cells under hypoxia. The 5 g/ml and 10 g/ml concentrations significantly promoted the proliferation of endothelial cells (P0.05).
CONCLUSION: 1. Endothelial progenitor cells with proliferative and differentiative potential can be successfully isolated from human peripheral blood by Ficoll method. The percentage of living cells is (96 65
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329
【参考文献】
相关期刊论文 前2条
1 李珊姗;杨镛;;Galectin-3对外周血内皮祖细胞源性血管内皮细胞增殖能力的影响[J];中国普外基础与临床杂志;2012年07期
2 杨国凯;杨镛;何晓明;罗开元;万嘉;杨光;李国剑;马震寰;陆平;;外周血干细胞移植治疗血栓闭塞性脉管炎[J];中国微创外科杂志;2009年09期
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