Foxc2过表达通过上调CXCR4增强内皮祖细胞促损伤血管内皮修复作用的研究

发布时间：2018-02-26 22:36

本文关键词： 内皮祖细胞 Foxc2 CXCR4 迁移粘附归巢募集再内皮化新生内膜增生　出处：《华中科技大学》2011年博士论文　论文类型：学位论文

【摘要】：内皮损伤或功能障碍是动脉粥样硬化和血管成形术后再狭窄等心血管疾病发生进展的始动环节。加快损伤内皮的修复可有效抑制新生内膜的增生,对动脉粥样硬化的早期预防和血管成形术后再狭窄的防治具有重要意义。近年的研究表明动员或移植的内皮祖细胞(endothelial progenitor cells, EPCs)可直接分化为内皮细胞,和或以旁分泌机制刺激成熟内皮增殖迁移,促进损伤血管的再内皮化,抑制新生内膜的增生。充足的归巢是移植EPCs功能发挥的前提,但大多数动物及临床实验显示移植EPCs的归巢和长期植入率都非常低,这也是导致治疗效果不理想的重要原因之一。事实上,EPCs在损伤区归巢聚集的数量不仅取决于循环中细胞数目也取决于移植细胞迁移粘附等生物学功能。有研究表明CXCR4的表达水平对EPCs的归巢及促内皮修复功能起重要的调控作用。然而,冠心病的危险因素可导致EPCs CXCR4表达下调或CXCR4的信号通路受损,导致EPCs的迁移归巢能力下降,严重影响了EPCs移植的疗效。因此,CXCR4表达及功能调控已成为EPCs治疗领域重要的研究课题。 Foxc2蛋白是叉头框转录因子家族成员之一,对于心血管系统起重要的调控作用。在胚胎发育期,Foxc2可以调控内皮基因表达及血管的发生。近来研究表明Foxc2可通过多个环节调控血管生成。例如,Foxc2可诱导多种粘附分子和促血管生成因子的表达。此外,Foxc2可直接调控内皮细胞CXCR4的表达。然而Foxc2对EPCs生物学特性的影响,迄今尚未见报道。基于此,我们欲探讨Foxc2对EPCs CXCR4表达、归巢功能及促内皮修复效应的影响。本研究分为三个部分。第一部分小鼠骨髓内皮祖细胞的培养及鉴定目的：分离培养小鼠骨髓源内皮祖细胞并鉴定。方法：分离小鼠骨髓单个核细胞,在内皮培养系定向诱导培养15-21天,通过形态学观察细胞的生长形态,用免疫荧光、Western blot及流式细胞仪技术检测内皮标记及造血系标记,Dil-acLDL摄取及Matrigel管样结构形成实验检测内皮分化的功能学特性。结果：分离骨髓单个核细胞诱导培养14天后,培养细胞逐渐呈现单层的“铺路石样”外观。免疫荧光检测结果显示,绝大多数细胞表达内皮标记物CD31、VE-cadherin及vWF; Western blot分析进一步证实培养细胞表达内皮标记FLK-1、CD31和VE-cadherin。而流式细胞仪分析显示培养细胞几乎不表达造血系标记CD34和CD45. Dil-acLDL摄取结果显示,约95%的细胞吸收Dil-acLDL。Matrigel管状结构形成实验显示培养细胞能围成管状、网络状结构。因此,从形态学、细胞表型及功能学鉴定来看,培养细胞具有内皮集落形成细胞(endothelial colony forming cells, ECFCs)或晚期EPCs特性。结论：小鼠骨髓单个核细胞在特定诱导扩增的培养条件下可获得EPCs。第二部分Foxc2过表达对内皮祖细胞迁移及粘附能力的影响目的：研究Foxc2过表达对EPCs体外迁移及粘附功能的影响及其机制。方法：将PcDNA3.1-Foxc2质粒用脂质体转染EPCs,转染48小时后用定量RT-PCR及Western blot检钡Foxc2基因的表达。用免疫荧光、Western blot、流式细胞仪及定量RT-PCR方法检测CXCR4的表达。用Transwell小室检测EPCs向SDF-1α迁移,用静止粘附实验检测EPCs与纤维连接蛋白的粘附。结果：定量RT-PCR及Western blot检测结果显示,PcDNA3.1-Foxc2质粒转染显著增加Foxc2 mRNA及蛋白的表达。免疫荧光、Western blot及流式细胞仪结果表明Foxc2过表达增加EPCs CXCR4蛋白的表达,定量RT-PCR检测显示Foxc2-EPCs CXCR4 mRNA水平大约是对照组的2倍(P＜0.05)。在SDF-1α刺激下,Foxc2-EPCs迁移及粘附能力明显高于Ctrl-EPCs,而AMD3100或LY294002可抑制Foxc2-EPCs增加的迁移及粘附效应。结论：Foxc2过表达能有效增加EPCs CXCR4的表达及体外迁移、粘附功能,Foxc2过表达所增强的迁移及粘附效应与CXCR4表达上调及下游PI3K/Akt信号活化增强有关。第三部分Foxc2过表达对内皮祖细胞归巢及促内皮修复作用的影响目的：研究Foxc2过表达对EPCs体内归巢及促内皮修复作用的影响。方法：建立小鼠颈动脉内膜损伤模型。从野生小鼠和GFP小鼠骨髓分离培养EPCs和GFP/EPCs,进行Foxc2质粒转染,再经尾静脉输注颈动脉内膜损伤小鼠体内,3天后观察损伤部位GFP标记细胞数目,以检测移植EPCs在体内归巢潜能；7天后用Evens蓝染色检测损伤血管再内皮化的程度,14天后观察GFP+内皮细胞的百分比,以检测移植GFP/EPCs掺入修复内皮层的情况,28天后组织学检测新生内膜和中膜面积的比值(N／M),评价EPCs移植抑制新生内膜增生的治疗效应。结果：GFP/EPCs移植3天后,GFP+严格局限于损伤部位的腔表面,Foxc2-GFP/EPCs移植组损伤血管募集的GFP+细胞明显多于Ctrl-GFP/EPCs组(约为Ctrl-GFP/EPCs组的2倍,P＜0.05)；7天后,Ctrl-EPCs移植组损伤血管再内皮化程度明显提高,Foxc2-EPCs组再内皮化程度又显著高于Ctrl-EPCs组(90.3±1.6%vs 57.2±1.3%,P＜0.05)；14天后,Foxc2-GFP/EPCs移植组GFP+内皮细胞百分比显著高于Ctrl-GFP/EPCs组(46.67±7.09%vs 31.50±5.26%,P＜0.05)；28天后,Ctrl-EPCs移植组小鼠损伤血管新生内膜的形成明显减少,N／M较PBS组降低了65%,而Foxc2-EPCs组新生内膜的增生程度降低更明显(N／M：0.38±0.03 vs 0.67±0.05,P＜0.05)。最后,Foxc2-EPCs移植前用AMD3100或LY294002预孵育能显著抑制Foxc2过表达所增强的EPCs归巢能力、促进再内皮化及抑制新生内膜增生的效应。结论：Foxc2过表达可促进EPCs在损伤内膜的归巢和募集,相应提高EPCs促内皮修复及抑制新生内膜增生的效应；而且Foxc2过表达增强的EPCs归巢及治疗效应与CXCR4/PI3K/Akt信号通路有关。
[Abstract]:Endothelial injury or dysfunction is the initiating factor in atherosclerosis and restenosis after angioplasty and other cardiovascular diseases. To accelerate the repair of endothelial injury can effectively inhibit neointimal hyperplasia, plays an important role in the prevention and early vascular angioplasty on atherosclerosis prevention after the narrow narrow again.
Recent studies suggest that mobilization or transplantation of endothelial progenitor cells (endothelial progenitor cells, EPCs) can directly differentiate into endothelial cells, and to stimulate or paracrine mechanism of mature endothelial proliferation and migration, promote reendothelialization, inhibition of neointimal hyperplasia. The homing sufficient is a prerequisite for transplantation of EPCs function, but most of the animal and clinical experiments showed that the homing EPCs transplantation and long-term implantation rate is very low, this is one of the important reasons causing the treatment effect is not ideal. In fact, EPCs in the damage zone homing aggregation number depends not only on the number of circulating cell transplantation depends on cell adhesion and migration and other biological functions. Studies have shown that expression of homing the level of CXCR4 and EPCs on promoting endothelial repair function plays an important role. However, the risk factors of coronary heart disease can lead to EPCs CXCR4 expression or CXCR4 letter The damage of pathway has led to the decrease of migration and homing ability of EPCs, which seriously affects the efficacy of EPCs transplantation. Therefore, CXCR4 expression and function regulation have become an important research topic in the field of EPCs treatment.
Foxc2 protein is a forkhead box transcription factor family, the cardiovascular system plays an important role. In the stage of embryonic development, Foxc2 can regulate endothelial gene expression and angiogenesis. Recent studies have shown that Foxc2 can regulate angiogenesis through multiple links. For example, Foxc2 can induce the expression of adhesion molecules and angiogenesis factor. In addition, the expression of Foxc2 can directly regulate endothelial cell CXCR4. The influence of Foxc2 on the biological characteristics of EPCs, however, has not been reported so far. Based on this, we want to explore the effect of Foxc2 on the expression of EPCs CXCR4, the homing function and the promotion of endothelial repair effect. This research is divided into three parts.
The culture and identification of bone marrow endothelial progenitor cells in the first part of mice
Objective: to isolate and culture the mouse bone marrow derived endothelial progenitor cells and identify them.
Methods: the isolation of bone marrow mononuclear cells, cultured and induced for 15-21 days of orientation in endothelial morphology was observed by morphology, immunofluorescence and Western blot detection of endothelial markers and flow cytometry hematopoietic markers, Dil-acLDL uptake and Matrigel tube formation assay function and endothelial differentiation characteristics.
Results: the separation of bone marrow mononuclear cells induced by cultured cells after 14 days of culture, gradually showing a cobblestone like appearance. Immunofluorescence showed that most cells express endothelial markers CD31, VE-cadherin and vWF; Western blot analysis further confirmed the cultured cells expressed endothelial markers FLK-1, CD31 and VE-cadherin. and flow cytometry. Instrument analysis showed that the cultured cells were almost not expressed hematopoietic markers CD34 and CD45. Dil-acLDL uptake showed that about 95% of the cells to absorb Dil-acLDL.Matrigel tube formation experiments showed that the cultured cells can form tubular network structure. Therefore, the morphology, phenotype and functional identification, cultured cells with endothelial colony forming cells (endothelial colony forming cells, ECFCs) or late EPCs characteristics.
Conclusion: the mouse bone marrow mononuclear cells can obtain EPCs. under specific induced culture conditions.
The effect of overexpression of Foxc2 on the migration and adhesion of endothelial progenitor cells in the second part
Objective: To study the effect of overexpression of Foxc2 on the migration and adhesion of EPCs in vitro and its mechanism.
Methods: PcDNA3.1-Foxc2 plasmid were transfected into EPCs, 48 hours after transfection with RT-PCR and Western blot expression quantitative detecting Foxc2 gene. By immunofluorescence, Western blot, flow cytometry and quantitative RT-PCR method to detect CXCR4. Detected by Transwell chamber EPCs to SDF-1 with alpha migration, adhesion of EPCs and fibronectin detection the static adhesion experiment.
Results: RT-PCR and Western quantitative blot assay showed that transfection of PcDNA3.1-Foxc2 plasmid significantly increased the expression of Foxc2 mRNA and protein. Immunofluorescence, Western blot and flow cytometry showed that Foxc2 overexpression increased the expression of EPCs CXCR4 protein, Foxc2-EPCs CXCR4 display quantitative RT-PCR detection of mRNA level is about 2 times that of the control group (P < 0.05). In SDF-1 stimulation, Foxc2-EPCs migration and adhesion ability was significantly higher than that of Ctrl-EPCs, migration and adhesion effect while AMD3100 or LY294002 can inhibit the Foxc2-EPCs increase.
Conclusion: Foxc2 overexpression can effectively increase EPCs CXCR4 expression and migration in vitro, and adhesion function. The enhanced migration and adhesion effect of Foxc2 overexpression is related to the up regulation of CXCR4 expression and the enhancement of downstream PI3K/Akt signal activation.
The effect of overexpression of Foxc2 on endothelial progenitor cell homing and endothelial repair in the third part
Objective: To study the effect of overexpression of Foxc2 on homing and endothelial repair in EPCs in vivo.
Methods: to establish a mouse model of carotid artery intimal injury. EPCs and GFP/EPCs were isolated and cultured from bone marrow of wild-type mice and GFP mice were transfected with Foxc2 plasmid, followed by intravenous injection of carotid intimal injury in mice was observed after 3 days, the number of injured parts of GFP labeled cells to detect the potential of EPCs transplantation in vivo homing; 7 days later by Evens the degree of blue staining detection of vascular endothelial injury, after 14 days of observation GFP+ percentage of endothelial cells, to detect transplanted GFP/EPCs incorporation repair endothelial layer, the ratio of detection of neointima and media area 28 days after the organization (N / M), to evaluate the curative effect of EPCs transplantation inhibits neointimal hyperplasia.
Results: GFP/EPCs 3 days after transplantation, cavity surface GFP+ strictly confined to the site of injury, injury of vascular Foxc2-GFP/EPCs transplantation group raised significantly more GFP+ cells than Ctrl-GFP/EPCs group (about 2 times that of the Ctrl-GFP/EPCs group P < 0.05); 7 days later, Ctrl-EPCs transplantation group reendothelialization significantly improved, Foxc2-EPCs group recellularization the degree was significantly higher than the Ctrl-EPCs group (90.3 + 57.2 1.6%vs + 1.3%, P < 0.05); 14 days later, Foxc2-GFP/EPCs transplantation group GFP+ endothelial cell percentage was significantly higher than that of Ctrl-GFP/EPCs group (46.67 + 31.50 7.09%vs + 5.26%, P < 0.05); 28 days later, the formation of Ctrl-EPCs mice of transplantation group neointimal / N decreased obviously. M was 65% lower than in PBS group, Foxc2-EPCs group and the degree of hyperplasia of neointima was reduced (N / M:0.38 + 0.03 vs 0.67 + 0.05, P < 0.05). Finally, using AMD3100 or LY294002 pre Foxc2-EPCs before transplantation Incubation can significantly inhibit the EPCs homing ability of Foxc2 over expression, promote re endothelialization and inhibit the effect of neointimal hyperplasia.
Conclusion: over expression of Foxc2 can promote the homing and recruitment of EPCs in injured intima, and enhance the effect of EPCs on endothelial repair and neointimal hyperplasia. Foxc2 overexpression enhances EPCs homing and its therapeutic effect is related to CXCR4/PI3K/Akt signaling pathway.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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