大鼠骨髓源性内皮祖细胞（EPCs）体外培养对人脐静脉内皮细胞（HUVEC）增殖的影响及黄芪多糖（APS）干预的研究

发布时间：2018-05-27 04:18

  本文选题：内皮祖细胞 + 黄芪多糖　； 参考：《贵阳中医学院》2015年硕士论文

【摘要】：目的：1.观察黄芪多糖(Astragalus Polysaccharides,APS)体外干预培养对大鼠骨髓源性内皮祖细胞(Endothelial Progenitor Cells,EPCs)数量、迁移、黏附、增殖、周期分布及分化的影响。2.观察APS体外干预大鼠骨髓源性EPCs共培养对人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells,HUVEC)增殖的影响。方法：1.取雄性Wistar大鼠予以水合氯醛腹腔注射麻醉,脱颈处死,将其股骨、胫骨、胸骨、肱骨取出,用PBS缓冲液冲洗骨髓腔,采用密度梯度离心法分离大鼠骨髓源性单个核细胞,培养7天以后收集贴壁细胞。采用多波长激光共聚焦显微镜对贴壁细胞进行鉴定,采用流式细胞术检测贴壁细胞的表型。收集大鼠骨髓源性EPCs,将其随机分为6组：设立对照组；其他组为APS各浓度组(共5组),于DMEM(低糖)培养基中加入终浓度为0.05mg/ml、0.1mg/ml、0.2mg/ml、 0.4mg/ml、0.8mg/ml的APS培养24h,其中APS浓度为0.4mg/ml组予以不同时间(Oh、6h、12h、24h、48h)进行培养。倒置显微镜观察每组大鼠骨髓源性EPCs并进行计数；Transwell小室培养检测大鼠骨髓源性EPCs的迁移数量；黏附能力实验检测大鼠骨髓源性EPCs的黏附数量；MTT增殖实验检测大鼠骨髓源性EPCs的增殖；流式细胞术检测大鼠骨髓源性EPCs的细胞周期的分布和分化。2.分离培养大鼠骨髓源性EPCs,7天后收集贴壁细胞,取样进行染色和流式鉴定,待细胞表达符合标准后,建立Transwell共培养体系,设立对照组,将大鼠骨髓源性EPCs与HUVEC予以DMEM(低糖)培养基共培养,并予以APS各浓度(0.05mg/ml、0.1mg/ml、0.2mg/ml、0.4mg/ml、0.8mg/ml)干预共培养24h后,MTT增殖实验检测APS各浓度干预共培养后HUVEC的增殖；其中APS浓度为0.4mg/ml组予以干预共培养不同时间(Oh、6h、12h、24h、48h),MTT增殖实验检测共培养各时间点HUVEC的增殖。结果：(1)APS体外干预培养对大鼠骨髓源性EPCs数量的影响：APS能明显增加大鼠骨髓源性EPCs的数量,并且呈一定的量效和时效关系,APS浓度为0. Olmg/ml时,其数量较对照组开始增加(P0.05),APS浓度为0.4mg/ml时达到最佳效应(P0.01)；APS浓度为0.4mg/ml干预培养12h后,其数量明显增加(P0.01),24h达到最佳效应(P0.01),48h稍有下降,但仍然优于对照组(P0.01)。(2)APS体外干预培养对大鼠骨髓源性EPCs迁移能力的影响：APS能明显提高大鼠骨髓源性EPCs的迁移能力,并且呈一定的量效和时效关系,AAPS浓度为0.1mg/ml时,其迁移数量较对照组开始增加(P0.05),APS浓度为0.4mg/ml时达到最佳效应(P0.01);APS浓度为0.4mg/ml干预培养6h后,其迁移数量开始增加(P0.05),24h达到最佳效应(P0.01),48h稍有下降,但仍然优于对照组(P0.01)。(3)APS体外干预培养对大鼠骨髓源性EPCs增殖能力的影响：APS能明显提高大鼠骨髓源性EPCs增殖的能力,并且呈一定的量效和时效关系,APS浓度为0.05mg/ml时,其增殖能力较对照组开始增加(P0.05),APS浓度为0.4mg/ml时达到最佳效应(P0.01);APS浓度为0.4mg/ml干预培养6h后,其增殖能力开始增加(P0.05),24h达到最佳效应(P0.01),48h稍有下降,但仍然优于对照组(P0.01)。(4) APS体外干预培养对大鼠骨髓源性EPCs周期分布的影响：APS浓度为0.1mg/ml时,G0/G1期大鼠骨髓源性EPCs的比例开始减少(P0.05),而S期和G2期的大鼠骨髓源性EPCs比例开始增加(P0.05),APS浓度为0.4mg/ml时达最佳效应(P0.01)。(5) APS体外干预培养对大鼠骨髓源性EPCs分化的影响：APS浓度为0.2mg/ml时开始促进大鼠骨髓源性EPCs向内皮细胞系方向分化,其表达单核/巨噬细胞表而标志(CD14+和CD64+)细胞百分比与对照组相比降低(P0.05或P0.01),而内皮细胞特异性标志(vWF+)细胞百分比与对照组相比升高(P0.05),APS浓度为0.4mg/ml时达最佳效应(P0.01)。(6)Transwell间接共同培养条件下APS干预大鼠骨髓源性EPCs对HUVEC增殖能力的影响：在APS不同浓度和不同时间体外干预下呈一定的量效和时效关系。APS体外干预培养HUVEC与对照组相比HUVEC增殖能力明显增加(P0.01)；大鼠骨髓源性EPCs和HUVEC共培养与对照组相比HUVEC增殖能力增加(P0.01);APS体外干预大鼠骨髓源性EPCs和HUVEC共培养与APS体外干预培养HUVEC相比HUVEC增殖能力明显增加(P0.01)。APS (0.4mg/ml)干预大鼠骨髓源性EPCs共培养不同时间与对照组相比HUVEC增殖能力增加(P0.05或P0.01),而且呈一定的时间依赖性,于24h达到最佳效应。结论：(1)APS体外干预大鼠骨髓源性EPCs能增加其数量,促进其迁移、黏附、增殖,且呈一定的浓度及时间依赖性。(2)APS体外干预大鼠骨髓源性EPCs能使其G0/G1期的细胞比例减少,而S期和G2期细胞比例增加。(3)APS体外干预大鼠骨髓源性EPCs能使其表达的单核/巨噬细胞表而标志(CD14+、CD64+)细胞百分比降低,内皮细胞特异性标志(vWF+)细胞百分比明显升高。(4)APS体外干预大鼠骨髓源性EPCs与HUVEC共培养能明显促进HUVEC的增殖,且呈一定的浓度及时间依赖性变化。
[Abstract]:Objective: 1. to observe the effect of Astragalus Polysaccharides (APS) on the quantity, migration, adhesion, proliferation, periodic distribution and differentiation of Endothelial Progenitor Cells (EPCs) in vitro, and the effect of.2. observation on human umbilical vein endothelial cells (Human Um) in vitro (Human Um) in vitro. The effects of the proliferation of bilical Vein Endothelial Cells, HUVEC). Methods: 1. the male Wistar rats were injected with chloral hydrate in the abdominal cavity, and the femur, tibia, sternum, humerus were removed, and the bone marrow cavity was washed with PBS buffer, and the bone marrow derived mononuclear cells were separated by density gradient centrifugation and collected for 7 days after culture. Mural cells were identified by multi wavelength laser confocal microscopy. The phenotype of adherent cells was detected by flow cytometry. The rat bone marrow derived EPCs was collected and divided into 6 groups randomly: the control group was set up; the other groups were APS concentration groups (5 groups), and the final concentration in the DMEM (low sugar) medium was 0.05mg/ml, 0.1mg/ml, The APS of 0.2mg/ml, 0.4mg/ml and 0.8mg/ml was cultured in 24h, in which the concentration of APS was cultured in group 0.4mg/ml (Oh, 6h, 12h, 24h, 48h). The bone marrow origin of rats in each group was observed and counted by inverted microscope. The adhesion quantity of PCs; MTT proliferation test to detect the proliferation of rat bone marrow derived EPCs; flow cytometry to detect the distribution of cell cycle of bone marrow derived EPCs in rats and differentiation and differentiation of.2. to culture rat bone marrow derived EPCs. 7 days later, the adherent cells were collected and sampled for staining and flow identification. After the cell expression was conformed to the standard, a common Transwell was established. The rat bone marrow derived EPCs and HUVEC were co cultured with DMEM (low sugar) medium, and the concentration of APS (0.05mg/ml, 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml) was co cultured for 24h, and MTT proliferation experiment was conducted to detect the proliferation of APS each concentration. A total of different time (Oh, 6h, 12h, 24h, 48h) and MTT proliferation test were used to detect the proliferation of HUVEC in each time point. Results: (1) the effect of APS in vitro intervention on the number of bone marrow derived EPCs in rats: APS can significantly increase the number of bone marrow derived EPCs in rats, and a certain dose effect and aging relationship, when APS concentration is 0. As compared with the control group (P0.05), the optimal effect (P0.01) was reached when the concentration of APS was 0.4mg/ml; APS concentration increased significantly (P0.01) after 0.4mg/ml intervention for 12h, 24h reached the best effect (P0.01), and 48h had a slight decrease, but it was still better than the control group (P0.01). (2) the effect of in vitro intervention on the ability of bone marrow derived migration in rats APS could significantly improve the migration ability of rat bone marrow derived EPCs, and showed a certain dose effect and aging relationship. When the concentration of AAPS was 0.1mg/ml, the number of migration began to increase compared with the control group (P0.05). The APS concentration was 0.4mg/ml when the concentration was 0.4mg/ml, and the concentration of APS was 0.4mg /ml, and the number of migration began to increase. To the best effect (P0.01), 48h was slightly decreased, but still better than the control group (P0.01). (3) the effect of APS in vitro intervention on the proliferation of bone marrow derived EPCs in rats: APS could significantly improve the ability of rat bone marrow derived EPCs proliferation, and showed a certain dose effect and aging system. When APS concentration was 0.05mg/ml, its proliferation ability began to begin with the control group. Increase (P0.05), APS concentration was 0.4mg/ml to reach the best effect (P0.01); APS concentration was 0.4mg/ml intervention culture 6h, the proliferation ability began to increase (P0.05), 24h reached the best effect (P0.01), 48h slightly decreased, but still better than the control group (P0.01). (4) At 0.1mg/ml, the proportion of bone marrow derived EPCs in G0/G1 rats began to decrease (P0.05), while the proportion of bone marrow derived EPCs in S and G2 rats began to increase (P0.05), and APS concentration was 0.4mg/ml. (P0.01). (5) the effect of intervention on bone marrow derived differentiation of rats in vitro The source EPCs differentiated into the endothelial cell line, and the percentage of the expression of mononuclear / macrophage markers (CD14+ and CD64+) decreased (P0.05 or P0.01) compared with the control group (P0.05 or P0.01), while the percentage of endothelial cell specific markers (vWF+) increased (P0.05) and APS concentration was 0.4mg/ml (P0.01). (6) Transwell indirect The effect of APS intervention on the proliferation of HUVEC in rat bone marrow EPCs under common culture conditions: a certain dose effect and aging relationship under the intervention of different concentrations and different times of APS in vitro.APS increased the proliferation ability of HUVEC in vitro compared with the control group (P0.01), and the co culture and control of rat bone marrow derived EPCs and HUVEC were compared with the control group (P0.01). The proliferation ability of the group was increased compared with the HUVEC (P0.01), and the proliferation ability of the bone marrow derived EPCs and HUVEC in the rat in vitro was significantly increased (P0.01).APS (P0.01).APS (0.4mg/ml), compared with the APS in vitro, and the proliferation ability of the bone marrow derived EPCs co culture of rats was increased at different times. The time dependence of 24h reached the best effect. Conclusion: (1) in vitro, APS intervention in rat bone marrow derived EPCs can increase its number, promote its migration, adhesion, proliferation, and have a certain concentration and time dependence. (2) APS in vitro intervention in rat bone marrow derived EPCs can reduce the proportion of cells in the G0/ G1 phase, while the proportion of S phase and G2 phase increases. 3) APS in vitro intervention in rat bone marrow derived EPCs could reduce the percentage of the expression of mononuclear / macrophage (CD14+, CD64+) cells and increase the percentage of endothelial cell specific markers (vWF+) cells. (4) APS in vitro intervention in rat bone marrow derived EPCs and HUVEC co culture can significantly promote the proliferation of HUVEC, and present a certain concentration in time. Inter dependent change.
【学位授予单位】：贵阳中医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

【相似文献】

相关期刊论文 前10条

1 李然;方靖琴;陈晓;郭宇;艾华;张伟国;;EPCs示踪大鼠脑原位胶质瘤的动态MRI研究[J];磁共振成像;2014年04期

2 王雪梅;邓明明;;EPCs与肝硬化门脉高压症的关系及其治疗展望[J];实用医学杂志;2012年24期

3 Xin-Peng Cong;Wen-Hui Wang;Xi Zhu;Can Jin;Liang Liu;Xin-Min Li;;Silence of STIM1 attenua=tes the proliferation and migration of EPCs after vascular injury and its mechanism[J];Asian Pacific Journal of Tropical Medicine;2014年05期

4 刘超;郭朋举;李圣博;姚星星;焦周阳;文冰;许华山;赵文增;;Combined Use of Transmyocardial Laser Revascularization and Endothelial Progenitor Cells Enhances Neovascularization and Regional Contractility in a Canine Model of Ischemic Hearts[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2014年02期

5 方立;陈美芳;余国龙;肖智林;陈晓彬;谢秀梅;;内皮祖细胞对血管平滑肌细胞增殖的影响[J];中南大学学报(医学版);2010年01期

6 陈琴;黄铭涵;江时森;徐标;;丹参多酚酸盐修复糖基化终产物引起的晚期EPCs功能障碍及其分子机制[J];中国中西医结合杂志;2010年06期

7 ;A novel vWF A3-GPI modified EPCs to enhance its adhesion ability to collegen[J];Journal of Medical Colleges of PLA;2011年04期

8 李巧;冉海涛;王志刚;钟世根;凌智瑜;李攀;李兴升;陈庆伟;;超声微泡联合Ad-EGFP/HIF-1α介导EPCs归巢大鼠缺血心肌[J];中国医学影像技术;2010年11期

9 常柏;李春深;;EPCs移植治疗DR的研究新进展[J];实用糖尿病杂志;2012年04期

10 王航;尹扬光;李巍;邓梦杨;康华丽;秦浙学;王强;卢巍;刘洁;黄岚;;过表达PDGFR-β的EPCs调控血管不良重构[J];中国病理生理杂志;2012年11期

相关会议论文 前10条

1 Alex F.Chen;;GTPCH I Overexpression Ameliorates Impaired EPCs Function in Type 1 Diabetes[A];中国药理学会第十次全国学术会议专刊[C];2009年

2 崔斌;黄岚;;eNOS基因修饰EPCs移植抑制损伤血管过度增殖改善血管内皮功能[A];第十三次全国心血管病学术会议论文集[C];2011年

3 ;Dynamic Changes and Significance of Circulating EPCs、VWF and TM in Patients with Traumatic Brain Injury[A];2011中华医学会神经外科学学术会议论文汇编[C];2011年

4 冉海涛;李巧;王志刚;钟世根;凌智瑜;李攀;李兴升;陈庆伟;;超声微泡联合Ad-EGFP/HIF-1α介导EPCs归巢大鼠缺血心肌的实验研究[A];第十届全国超声心动图学术会议论文[C];2010年

5 张晓芸;崔晓栋;官秀梅;李宏;成敏;;稳定细胞骨架对EPCs功能的影响[A];中国生理学会第九届全国青年生理学工作者学术会议论文摘要[C];2011年

6 李巧;李兴升;王志刚;冉海涛;钟世根;凌智瑜;李攀;陈庆伟;;超声微泡联合Ad-EGFP/HIF-1α介导EPCs移植治疗大鼠心肌梗死的研究[A];第十届全国超声心动图学术会议论文[C];2010年

7 何智辉;;5-杂氮-2‘-脱氧胞苷对CSES所致EPCs功能及其Sca-1表达的影响[A];中华医学会呼吸病学年会——2013第十四次全国呼吸病学学术会议论文汇编[C];2013年

8 杨瑞瑞;杜怡峰;郭守刚;;VEGF动员内源性EPCs促进MCAO/R小鼠神经功能修复[A];山东省2013年神经内科学学术会议暨中国神经免疫大会2013论文汇编[C];2013年

9 霍亚南;黎金凤;;SDF-1对糖尿病患者外周血EPCs功能影响的研究[A];江西省中西医结合学会内分泌专业委员会第二次学术会议、内分泌与代谢性疾病中西医结合诊治新进展学习班资料汇编[C];2013年

10 崔晓栋;张晓芸;李宏;官秀梅;李鑫;成敏;;流体剪切应力对EPCs生物学特性的影响[A];第十届全国生物力学学术会议暨第十二届全国生物流变学学术会议论文摘要汇编[C];2012年

相关重要报纸文章 前1条

1 民泾;EPCs捕获技术改善支架生物相容性[N];医药经济报;2003年

相关博士学位论文 前10条

1 Shah Ourban Ali;家禽EPCs分离培养及HGF对炎症环境下EPCs功能的影响[D];浙江大学;2015年

2 尉辉杰;阿托伐他汀动员EPCs延缓颅内动脉瘤发展的机制及降低栓塞后复发的潜在价值[D];天津医科大学;2016年

3 叶家欣;强化他汀治疗促进EPCs动员改善心肌缺血的临床和基础研究[D];南京医科大学;2012年

4 陈飞兰;内皮祖细胞（EPCs）在胶质瘤微血管构筑表型异质性形成中的作用及其机制研究[D];第三军医大学;2008年

5 杨兆华;内皮祖细胞（EPCs）对移植性动脉硬化的作用及其机制研究[D];复旦大学;2009年

6 王航;过表达PDGFR-β的EPCs在损伤血管修复中作用研究[D];第三军医大学;2012年

7 崔斌;eNOS基因修饰EPCs移植与损伤内膜修复的实验研究[D];第三军医大学;2008年

8 谭虎;vWF A3-GPI强化EPCs富集及其在损伤血管壁修复中作用的实验研究[D];第三军医大学;2008年

9 况春燕;STIM1/TRPC1复合体对EPCs修复损伤血管能力的影响[D];第三军医大学;2011年

10 喻杨;血管损伤修复中CCNI对EPCs功能的影响及其机制研究[D];第三军医大学;2009年

相关硕士学位论文 前10条

1 孙丽娜;Visfatin对内皮祖细胞凋亡的影响及其机制探讨[D];河北医科大学;2015年

2 薛国能;用于捕获和归巢EPCs的Fucoidan与CD133抗体/SDF-1复合生物涂层的构建[D];西南交通大学;2015年

3 南伟;不同来源的EPCs对NSCs增殖分化的影响[D];兰州大学;2016年

4 郑淑欣;负载骨髓MSCs和/或EPCs的羊膜支架修复犬长段尿道缺损的研究[D];西北农林科技大学;2016年

5 陈晨;犬骨髓EPCs的分离、鉴定和标记及组织工程化尿道的构建[D];西北农林科技大学;2016年

6 郭军;mICAM-1在MSCs和EPCs间黏附作用的研究[D];石河子大学;2016年

7 王照飞;CXCR4过表达HUCB-late EPCs移植对后肢缺血组织血管新生的实验研究[D];湖南师范大学;2016年

8 刘欢;EPCs与BMSCs复合细胞膜片促进放疗区种植体骨结合的实验研究[D];第四军医大学;2016年

9 朱耀;桃红四物汤对大鼠创伤性股骨头缺血坏死模型外周血中EPCs表达影响的研究[D];湖南中医药大学;2016年

10 陈春媛;人EPCs来源外泌体修复糖尿病大鼠皮肤缺损的作用及机制[D];南昌大学;2016年

，

本文编号：1940389