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类型 基础研究 预答辩日期 2018-01-08
开始(开题)日期 2015-01-26 论文结束日期 2017-09-30
地点 东南大学附属中大医院2号楼5楼ICU医生办公室 论文选题来源 国家自然科学基金项目     论文字数 4.4 (万字)
题目 Ang II-AT2R介导间充质干细胞向急性肺损伤小鼠肺组织归巢的机制和效应研究
主题词 间充质干细胞,急性肺损伤,血管紧张素II,血管紧张素2型受体,细胞迁移
摘要 第一部分 Ang II-AT2R通过FAK和RhoA/Cdc42信号途径促进间充质干细胞的迁移 目的:间充质干细胞(Mesenchymal stem cells,MSCs)在通过血液迁移到损伤部位过程中可能受到炎症介质的趋化。研究表明促炎症介质血管紧张素II(Angiotensin II,Ang II)在体外可以通过血管紧张素II受体介导促进多种类型细胞的迁移,但Ang II对MSCs迁移的影响及其作用机制尚不明确。 方法:(1) 使用RT-PCR法检测MSCs表面血管紧张素II受体(AT1R和AT2R)mRNA的表达情况。(2) 将人源骨髓间充质干细胞进行常规体外培养,然后使用不同浓度(10-8 M, 10-7 M, 10-6 M, 10-5 M and 3×10-5 M)Ang II进行刺激,明确Ang II促进MSCs迁移的最佳刺激浓度后,再联合AT1R拮抗剂(Losartan,5μM)和/或AT2R拮抗剂(PD-123319,5μM)预处理后,使用划痕实验和Transwell小室迁移测定法评估MSCs的迁移能力。(3) 使用MTT法检测不同浓度Ang II对MSCs增殖能力的影响。(4) 使用粘着斑激酶(focal adhesion kinase, FAK)抑制剂PF-573228,RhoA抑制剂C3转移酶,Rac1抑制剂NSC23766或Cdc42抑制剂ML141来研究细胞粘附蛋白和Rho-GTPase蛋白家族(RhoA,Rac1和Cdc42)在Ang II介导MSCs迁移中的作用。(5) 通过western blot法检测细胞粘附蛋白(FAK,Talin和Vinculin)的表达。(6) 通过G-LISA法检测Rho-GTP酶家族蛋白活性。(7) 使用免疫荧光法评估反映肌动蛋白细胞骨架组织的F-肌动蛋白的水平。 结果:(1) 人源骨髓MSCs表达Ang II的两种受体AT1R和AT2R。(2) Ang II促进MSCs迁移的最佳浓度为10-7 M(P<0.05),且阻断AT2R后可明显抑制Ang II的促迁移作用(P<0.05),而阻断AT1R无法抑制Ang II的促迁移作用(P>0.05),并且随着浓度的增高,AT2R mRNA表达逐渐升高,在10-7 M浓度下表达水平最高,共同表明Ang II主要通过AT2R促进MSCs的迁移。(3) 相同浓度的Ang II对MSCs的增殖能力无明显促进作用(P>0.05),表明增强的MSCs迁移不是由Ang II促进的细胞增殖所介导的。(4) 阻断FAK,RhoA,Cdc42后,Ang II增强的MSCs迁移被明显抑制(P<0.05),而阻断Rac 1无法抑制Ang II的促迁移作用(P>0.05)。(5) 黏着斑蛋白激酶FAK激活对于黏着斑的形成至关重要,阻断FAK后,可见黏着斑的关键蛋白Talin和Vinculin表达明显被抑制(P<0.05)(6) Ang II刺激后可明显促进细胞骨架重排,而阻断FAK,RhoA,Cdc42后细胞骨架重排被显著抑制,而阻断Rac 1对Ang II的促细胞骨架重排无明显影响。并且阻断FAK后可明显抑制RhoA和Cdc42的活性(P<0.05),而对Rac 1的活性无明显影响。共同表明Ang II 激活FAK后促进RhoA和Cdc42激活从而增加细胞骨架重排,进而促进细胞收缩,共同影响MSCs的迁移。(7) 阻断AT2R可阻止Ang II刺激MSCs后FAK,Talin和Vinculin表达增高(P<0.05)以及F-肌动蛋白重组,表明Ang II主要通过AT2R活化FAK和RhoA/Cdc42从而介导黏着斑的形成以及F-肌动蛋白重组。 结论:Ang II-AT2R通过FAK和RhoA/Cdc42途径的信号传导调节人骨髓MSCs迁移。 第二部分 高表达AT2R的MSC向急性肺损伤小鼠肺组织的归巢及其肺保护效应的研究 目的:明确过表达AT2R的MSCs移植入急性肺损伤小鼠体内对其向损伤肺组织的迁移以及对肺损伤修复的影响。 方法:构建人过表达AT2R的慢病毒载体,然后转导入人源骨髓MSCs。同时还使用携带AT2R shRNA的慢病毒载体转导MSCs下调AT2R mRNA表达。(1) 使用荧光显微镜检测GFP的表达来验证转染效率和检测AT2R基因的表达情况来验证转染效果。(2) 体外使用Transwell小室迁移测定法检测调节AT2R表达后MSCs迁移能力。细胞准备稳妥后,使用气道内滴入LPS复制ALI小鼠模型,造模4h后按分组给予不同处理,24h和72h后。 (3) 近红外(Near-infrared,NIR)离体器官(肺脏)成像、肺组织荧光镜检及检测人特异性序列Alu的表达评价AT2R对MSCs在损伤肺组织内靶向归巢作用的影响。(4) 计算肺湿重/体重比评价肺水肿程度;伊文思蓝漏出实验评价肺微血管内皮通透性;评价AT2R对MSCs在ALI小鼠肺微血管内皮细胞的修复作用的影响。(5) H&E染色行组织病理学检查并进行肺损伤评分以评价肺损伤严重程度;留取肺泡灌洗液(Bronchoalveolar lavage fluid,BALF),计数肺泡灌洗液中有核细胞总数和瑞氏染色进行中性粒细胞分类计数评价炎症细胞的浸润程度;ELISA(Enzyme linked immunosorbent assay)法检测BALF中IL-1β、IL-6、IL-10的浓度评价肺部炎症反应的强度,从而综合评价AT2R对MSCs在ALI小鼠肺组织失控炎症反应的抑制效应和肺损伤修复作用的影响。 结果:(1) 慢病毒介导高表达以及干扰AT2R的MSCs,荧光显微镜观察转染后转染效率高达90%以上。与未转染的MSCs相比,AT2R高表达的MSCs(MSC-AT2R)其AT2R mRNA水平是未转染细胞的约3倍(P<0.05),而AT2R干扰的MSCs(MSC-ShAT2R)其AT2R mRNA水平较未转染细胞表达降低约80%(P<0.05)。(2) 体外实验中,与过表达对照组MSC-GFP相比,MSC-AT2R组Ang II促进人骨髓MSCs迁移的作用增强(P<0.05)。与干扰对照组MSC-Shcontrol相比,MSC-ShAT2R可抑制Ang II的促迁移作用(P<0.05)。(3) 离体器官成像结果显示,与MSC-GFP相比,MSC-AT2R组移植后24h和72h在肺组织内的荧光信号明显增多,而与MSC-Shcontrol相比,MSC-ShAT2R组移植后肺组织内的荧光信号明显降低;肺组织荧光镜检以及检测Alu水平也可见类似的效果,MSC-AT2R组移植后肺组织表达绿色荧光蛋白以及Alu序列显著增多(P<0.05),而MSC-ShAT2R组移植后肺组织表达绿色荧光蛋白以及Alu序列显著减少,表明MSC-AT2R组移植后在肺组织存留的数目明显增加,而MSC-ShAT2R组移植后肺组织存留的数目明显减少。(4) MSC-AT2R组能更显著的减少肺湿重/体重比、降低肺微血管内皮对伊文思蓝的通透性(P<0.05)。而MSC-ShAT2R组减少肺湿重/体重比、降低肺微血管内皮对伊文思蓝的通透性能力(P<0.05)。(5) MSC-AT2R治疗可显著减轻肺组织病理损伤、降低肺损伤评分(P<0.05),降低BALF中有核细胞总数、中性粒细胞数(P<0.05),IL-1β、IL-6水平降低(P<0.05),并增加IL-10水平(P<0.05)。反之,MSC-ShAT2R移植可加重LPS诱导的ALI小鼠肺病理损伤,增加肺损伤评分(P<0.05),使BALF中有核细胞总数、中性粒细胞数升高(P<0.05),IL-1β、IL-6水平升高(P<0.05),并降低IL-10水平(P<0.05)。 结论:过表达AT2R可显著增强MSCs的迁移能力并增加其在ALI损伤肺组织内的存留,从而更好的促进肺内屏障功能的恢复,抑制肺内的炎症反应,促进肺损伤的修复。
英文题目 Angiotensin II-AT2R Mediates Marrow Derived Mesenchymal Stem Cells homing to injured lung tissue following acute lung injury in a mice model
英文主题词 Mesenchymal stem cells, acute lung injury, Angiotensin II, AT1R, AT2R,cell migration, FAK, Rho GTPases, F-actin
英文摘要 Part One: Ang II-AT2R Increases Mesenchymal Stem Cell Migration by signaling through the FAK and RhoA/Cdc42 Pathways in vitro Objective: Mesenchymal stem cells (MSCs) migrate via the bloodstream to sites of injury and are possibly attracted by inflammatory factors. As a pro-inflammatory mediator, Angiotensin II (Ang II) reportedly enhances the migration of various cell types by signaling via the angiotensin II receptor in vitro. However, few studies have focused on the effects of Ang II on MSC migration and the underlying mechanisms. Methods: Human bone marrow MSC migration was measured using wound healing and Boyden chamber migration assays after treatments with different concentrations of Ang II, an AT1R antagonist (Losartan) and/or an AT2R antagonist (PD-123319). To exclude the effect of proliferation on MSC migration, we measured MSC proliferation after stimulation with the same concentration of Ang II. Additionally, we employed the focal adhesion kinase (FAK) inhibitor PF-573228, RhoA inhibitor C3 transferase, Rac1 inhibitor NSC23766 or Cdc42 inhibitor ML141 to investigate the role of cell adhesion proteins and the Rho-GTPase protein family (RhoA, Rac1, and Cdc42) in Ang II-mediated MSC migration. Cell adhesion proteins (FAK, Talin and Vinculin) were detected by western blot. The Rho-GTPase family protein activities were assessed by G-LISA and F-actin levels, which reflect actin cytoskeletal organization, were detected by using immunofluorescence. Results: Human bone marrow MSCs constitutively expressed AT1R and AT2R. Additionally, Ang II increased MSC migration in an AT2R-dependent manner. Notably, Ang II-enhanced migration was not mediated by Ang II-mediated cell proliferation. Interestingly, Ang II-enhanced migration was mediated by FAK activation, which was critical for the formation of focal contacts, as evidenced by increased Talin and Vinculin expression. Moreover, RhoA and Cdc42 were activated by FAK to increase cytoskeletal organization, thus promoting cell contraction. Furthermore, FAK, Talin and Vinculin activation and F-actin reorganization in response to Ang II were prevented by PD-123319 but not Losartan, indicating that FAK activation and F-actin reorganization were downstream of AT2R. Conclusions: These data indicate that Ang II-AT2R regulates human bone marrow MSC migration by signaling through the FAK and RhoA/Cdc42 pathways. This study provides insights into the mechanisms by which MSCs home to injury sites and will enable the rational design of targeted therapies to improve MSC engraftment. ? Part Two: Genetic Modification of Mesenchymal Stem Cells Overexpressing Angiotensin II Type 2 Receptor Increase Lung Engraftment in LPS-induced Acute Lung Injury Mice Objective: The objective of our study was to determine whether overexpression of AT2R in MSCs augments their cell migration and engraftment after systemic injection in ALI mice. Methods: A human AT2R expressing lentiviral vector was constructed and introduced into human bone marrow MSCs. We also down-regulated AT2R mRNA expression, using a lentivirus vector carrying AT2R shRNA to transfect MSCs. The effect of AT2R expression on migration of MSCs was examined in vitro. A mouse model of lipopolysaccharide induce ALI was used to investigate the engraftment of AT2R overexpression MSCs and the therapeutic potential in vivo. Results: (1) Lentivirus-mediated high expression and interference with AT2R MSCs, transfection efficiency was up to 90% by fluorescence microscopy. Compared with untransfected MSCs, the AT2R mRNA level of AT2R overexpressed MSC (MSC-AT2R) was about 3 times that of untransfected cells (P<0.05), while AT2R-interfering MSC (MSC-ShAT2R) showed less than 80% that of untransfected cells (P<0.05). The effect of Ang II on the migration of human bone marrow MSCs in MSC-AT2R group was significantly higher than that in control group (P<0.05). MSC-ShAT2R inhibited the migration induced by Ang II (P<0.05) compared with MSC-Shcontrol group. (3) Compared with MSC-GFP, the fluorescence signal of lung tissue in MSC-AT2R group was significantly increased at 24h and 72h after transplantation. Compared with MSC-Shcontrol, MSC-ShAT2R group (P<0.05), and the expression of green fluorescent protein and Alu sequence in the lung tissue of MSC-AT2R group was significantly increased (P<0.05), while the fluorescence intensity of the lung tissue was significantly decreased in the lung tissue fluorescence microscopy and the Alu level. These date indicated that the MSC retention of lung tissue in MSC-AT2R group were significantly increased after transplantation, while the MSCof retention lung tissue in MSC-ShAT2R group were significantly reduced. (P<0.05). The lung wet weight/body weight ratio was reduced and the permeability of pulmonary microvascular endothelium was decreased (P<0.05). While the MSC-ShAT2R group reduced the lung wet weight/body weight ratio and decreased the permeability of pulmonary microvascular endothelium (P<0.05). (5) MSC-AT2R treatment can significantly reduce the pathological damage of lung tissue, reduce lung injury score (P<0.05), reduce the total number of cells and neutrophils in BALF (P<0.05), reduce IL-1β, IL-6 levels (P<0.05), and increase IL-10 level (P<0.05). In contrast, MSC-ShAT2R transplantation could aggravate lung injury in LPS-induced lung injury and increase lung injury score (P<0.05), increase the number of cells and neutrophils in BALF (P<0.05), IL-1β, IL-6 levels (P <0.05), and decrease the level of IL-10 (P<0.05). Conclusions: Our results demonstrate that overexpression of AT2R enhance the migration and engraftment of MSCs and may provide a new therapeutic strategy for the injured lung.
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代表作
论文名称
Ang II-AT2R increases mesenchymal stem cell migration by signaling through the FAK and RhoA/Cdc42 pa
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
黄培林 正高 教授 博导 东南大学
侯百东 正高 教授 博导 中国科学院生物物理研究所
邓国民 正高 教授 博导 南京医科大学
张建琼 正高 教授 博导 东南大学医学院
杨毅 正高 教授 博导 东南大学附属中大医院
      
答辩秘书信息
姓名职称工作单位备注
谢剑锋 其他 主治医师 东南大学附属中大医院