碱性成纤维细胞生长因子对糖尿病内皮祖细胞作用研究

发布时间：2018-06-12 18:47

  本文选题：内皮祖细胞 + 骨髓　； 参考：《重庆医科大学》2015年硕士论文

【摘要】：糖尿病微血管病变已成为威胁人类健康的重要杀手。糖尿病病人拔牙创难以愈合是拔牙术后的主要并发症。血管化困难是糖尿病难愈性创面发生的重要原因。大量研究结果显示内皮祖细胞(endothelial progenitor cells, EPCs)在血管化中起着重要作用。EPCs是内皮细胞的前体细胞,不仅能直接分化为血管内皮细胞参与血管发生和修复,还可通过分泌细胞因子改善内皮功能,促进血管新生及创面愈合。碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)在创伤愈合及组织再生过程中起着重要作用,多用于口腔溃疡等创面的治疗。以往实验研究显示,bFGF可显著改善EPCs的体外增殖、迁移等功能。而bFGF是否对糖尿病损害的内皮祖细胞具有同样的作用,尚未见相关研究报道。因此本实验通过对糖尿病大鼠的内皮祖细胞体外分离培养,观察不同浓度的bFGF对糖尿病大鼠内皮祖细胞的作用,为利用bFGF和EPCs治疗糖尿病难愈创面提供理论及实验依据。本实验分为以下两个部分：第一部分大鼠骨髓和脾脏来源的内皮祖细胞体外分离、培养鉴定及功能检测目的：分别提取大鼠骨髓和脾脏来源的EPCs,比较其生物学特性,为下一步实验EPCs的组织来源选择提供指导。从大鼠骨髓和脾脏内皮祖细胞提取,比较其生物学特性,为下一步的来源EPCs提供指导。方法：采用密度梯度离心法,从健康4周龄SD大鼠,获取骨髓和脾脏的单个核细胞,培养7d后,采用细胞膜红色荧光标记的乙酰化低密度脂蛋白(DiI-ac-LDL)和绿色荧光标记的荆豆凝集素(FITC-UEA-1)染色贴壁细胞鉴定EPCs,对比分析两种EPCs的迁移、黏附和一氧化氮(nitric oxide, NO)分泌能力。结果：体外分离培养得到的骨髓和脾脏来源的EPCs细胞形态基本相似。绝大部分细胞吞噬DiI-ac-LDL和FITC-UEA-1双染为黄色。骨髓来源的EPCs在细胞增殖、迁移、黏附和NO分泌能力方面均显著高于脾脏来源的EPCs。结论：骨髓相对于脾脏,更适宜作为体外分离培养EPCs的组织来源。第二部分碱性成纤维细胞生长因子对糖尿病大鼠内皮祖细胞增殖、迁移及成血管作用研究目的：观察不同浓度bFGF对糖尿病大鼠骨髓EPCs体外增殖、迁移及成血管功能的影响,为利用bFGF和EPCs治疗糖尿病难愈创面提供理论及实验依据。方法：使用高糖饮食加链脲佐菌素(Streptozocin,STZ)注射的方法诱导形成Ⅱ型糖尿病大鼠模型。采用密度梯度离心法从糖尿病大鼠骨髓获得单个核细胞,培养7d后收集贴壁细胞,DiI-ac-LDL和FITC-UEA-1双吞实验鉴定EPCs。取第1代EPCs,加入不同浓度bFGF (12.5.25.50、100、200ng/mL)培养,MTT法和Matrigel基质胶,Transwell小室检测糖尿病内皮祖细胞的增殖,迁移和体外血管生成能力的变化。结果：Ⅱ型糖尿病大鼠骨髓来源的单个核细胞绝大部分吞噬DiI-ac-LDL和FITC-UEA-1双染为黄色,鉴定其为EPCs。糖尿病大鼠骨髓EPCs相对于正常大鼠EPCs,其增殖、迁移及体外成血管功能均明显下降。12.5、25、50、100、200ng/mLbFGF均能提高糖尿病大鼠EPCs的增殖和迁移能力,且促进糖尿病大鼠EPCs增殖的最佳浓度为25ng/mL;促进糖尿病大鼠EPCs迁移的最适浓度为50ng/mL。体外Matrigel基质胶上EPCs未形成明显管状结构。结论：Ⅱ型糖尿病大鼠EPCs的增殖、迁移及成血管功能较正常大鼠EPCs明显下降。糖尿病大鼠EPCs增殖和迁移能力在一定浓度的bFGF作用下能够提升,但对体外血管形成能力无明显作用。
[Abstract]:Diabetic microvascular lesions have become an important killer of human health. The difficult healing of diabetes patients is the main complication after extraction. The difficulty of vascularization is an important cause of diabetes refractory wound. A large number of research results show that endothelial progenitor cells (EPCs) plays a role in vascularization. The important role of.EPCs is the precursor cells of endothelial cells, which can not only directly differentiate into vascular endothelial cells to participate in angiogenesis and repair, but also improve endothelial function by secreting cytokines, promote angiogenesis and wound healing. Basic fibroblast growth factor (basic fibroblast growth factor, bFGF) reacts in wound healing and tissue re In the process of birth, it plays an important role in the treatment of wounds such as oral ulcers. Previous experimental studies have shown that bFGF can significantly improve the proliferation and migration of EPCs in vitro, and whether bFGF has the same effect on endothelial progenitor cells damaged by diabetes and has not yet been found in the related research. The progenitor cells were isolated and cultured in vitro to observe the effect of different concentrations of bFGF on the inner skin progenitor cells of diabetic rats and provide theoretical and experimental basis for the treatment of diabetic refractory wound surface using bFGF and EPCs. The experiment was divided into two parts: Part 1: isolation, culture identification and function of endothelial progenitor cells from bone marrow and spleen derived from the first part of rats Objective: to extract EPCs from the bone marrow and spleen of rats respectively, to compare their biological characteristics, and to provide guidance for the selection of the tissue source of EPCs in the next step. From the bone marrow and spleen endothelial progenitor cells of rats, the biological characteristics were compared, and the guidance for the next source of EPCs was provided. Method: density gradient centrifugation was used, from health 4. The mononuclear cells of bone marrow and spleen were obtained from SD rats of week age, and after 7d were cultured, EPCs was identified by acetylated low density lipoprotein (DiI-ac-LDL) and green fluorescent tagged agglutinin (FITC-UEA-1) stained adherent cells labeled with green fluorescence. The migration, adhesion and nitric oxide (nitric oxide, NO) secretion of two kinds of EPCs were compared and analyzed. Results: the form of EPCs cells derived from bone marrow and spleen in vitro was basically similar. Most of the cells phagocytic DiI-ac-LDL and FITC-UEA-1 double dye were yellow. The bone marrow derived EPCs was significantly higher in cell proliferation, migration, adhesion and NO secretion than that of the spleen from the EPCs. conclusion: bone marrow is more than the spleen. Suitable as a tissue source for in vitro isolation and culture of EPCs. Second basic fibroblast growth factor (BBF) on the proliferation, migration and angiogenesis of inner skin progenitor cells in diabetic rats: the effects of different concentrations of bFGF on the proliferation, migration and vascular function of bone marrow EPCs in diabetic rats, for the treatment of bFGF and EPCs The theoretical and experimental basis of diabetic wound healing was provided. Methods: the model of type II diabetic rats was induced by high glucose diet plus Streptozocin (STZ) injection. The density gradient centrifugation was used to obtain mononuclear cells from the bone marrow of diabetic rats, and 7d was collected to collect parietal cells, DiI-ac-LDL and FITC-UEA-1 double swallowing. EPCs. was tested for first generation of EPCs, adding different concentrations of bFGF (12.5.25.50100200ng/mL), MTT and Matrigel matrix, Transwell chamber to detect the proliferation, migration and angiogenesis of diabetic endothelial progenitor cells. Results: most of the mononuclear cells derived from bone marrow of type II diabetic rats were phagocytic DiI-ac-LD The double staining of L and FITC-UEA-1 was yellow. It was identified that the bone marrow EPCs of EPCs. diabetic rats was EPCs, its proliferation, migration and in vitro vascular function decreased obviously.12.5,25,50100200ng/mLbFGF can increase the proliferation and migration ability of EPCs in diabetic rats, and the optimum concentration of EPCs in diabetic rats was 25ng/mL. The optimum concentration of EPCs migration in diabetic rats was that EPCs did not form an obvious tubular structure on the 50ng/mL. Matrigel matrix gel. Conclusion: the proliferation, migration and vascular function of EPCs in type II diabetic rats were significantly lower than that of normal rats. The proliferation and migration energy of EPCs in diabetic rats could be enhanced under the action of bFGF in a certain concentration. However, there was no significant effect on the ability of angiogenesis in vitro.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.1

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