经血来源的间充质干细胞（MenSCs）在急性肺损伤（ALI）治疗中作用的研究

发布时间：2018-02-25 09:25

本文关键词： 间充质干细胞 MenSCs 急性肺损伤细胞治疗　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：间充质干细胞是一种来源于中胚层的成体干细胞,具有自我更新和多向分化潜能等特点,可以在再生医学领域发挥重要的作用。经血来源的间充质干细胞(MenSCs)是间充质干细胞中的一员,主要从女性月经血中提取,具有获取方便,安全无毒副作用等优点。相较于其他来源的间充质干细胞,MenSCs没有伦理及创伤方面的困扰,且能不断从同一志愿者获得大量相同遗传背景的细胞。目前,MenSCs已经被用于心肌梗死、I型糖尿病等多种疾病的治疗研究。许多感染性疾病如H7N9,败血症及一些细菌尤其是Gram-的感染均可以引起急性肺损伤,然而,目前尚未发现MenSCs在急性肺损伤(ALI)方面的应用。本课题主要研究MenSCs在ALI的生物治疗中的作用。在本研究中,我们首先对MenSCs的进行了验证,发现分离的细胞呈梭状,表达 CD29,CD44,CD73,CD90,CD105,不表达 CD34,CD45 以及 CD11b,CD14,CD19,CD79a,CD117和HLA-DR分子,符合国际细胞治疗学会制定的MSCs标准,为MSCs中的一员。同时我们证实了 MenSCs具有良好的体外增殖能力,可以进行成骨成脂分化,具有多向分化能力。此外,我们还发现MenSCs可以表达CD140b,CD194,CD221,及CD184等趋化因子或者具有趋化能力的相关因子的受体,可以在多种细胞因子的刺激下进行迁移,发挥靶向治疗的作用。接着,我们探讨了 MenSCs在治疗ALI中的作用。在体外实验中,我们发现MenSCs可以向暴露于LPS的肺支气管上皮细胞BEAS-2B迁移,并保护其活性,减少细胞凋亡。同时,我们在体外对MenSCs进行了诱导,发现诱导后的细胞成扁平的上皮样形状,并表达SPB,SPC和SPD等肺表面活性蛋白。在LPS诱导的ALI模型小鼠体内,我们用X线和病理组织切片证实MenSCs可以缓解小鼠肺部炎症症状,减少损伤。而肺干/湿比和β-catenin蛋白表达的升高及支气管肺泡灌洗液(BALF)总蛋白的下降则提示着MenSCs移植后小鼠肺通透性的改善。我们对BALF中炎性细胞,MPO检测进一步验证了 MenSCs抑制肺部炎症的作用。同时,我们还发现MenSCs可以提高PCNA的表达量,减少Caspase-3的表达量,促进损伤肺的修复。最后,我们探讨了 MenSCs治疗急性肺损伤的可能机制。我们发现,在MenSCs移植后,小鼠BALF,血清及肺实质中抑炎症因子IL-10,TGF-β表达升高,促炎症因子IL-1β和IL-6表达降低,表明MenSCs可能是通过免疫调节的作用来抑制炎症。同时,我们检测了肺实质中KGF及VE-cadherin,β-catenin的表达,发现其表达量在MenSCs移植后均升高,表明MenSCs可以促进黏附蛋白的表达,形成黏附连接,促进细胞增生和修复。我们用WB检测了肺组织中PI3K,p-gsk3β,gsk3β,p-Src和Src蛋白的表达量,发现PI3K,gsk3β/β-catenin及Src可能参与了肺的修复。这些结果表明MenSCs可以在LPS介导的小鼠急性肺损伤中发挥治疗作用。
[Abstract]:Mesenchymal stem cells (MSCs) are adult stem cells derived from mesoderm and have the characteristics of self-renewal and multi-differentiation potential. Mesenchymal stem cells (Mens), which are derived from blood, are one of the mesenchymal stem cells, which are extracted mainly from the menstrual blood of women. Compared with other mesenchymal stem cells, MenSCs have no ethical or traumatic problems. At present, MenSCs have been used in the treatment of many diseases such as myocardial infarction and type I diabetes. Many infectious diseases such as H7N9, septicemia and some bacteria. It's the Gram- infection that can cause acute lung injury, However, the application of MenSCs in acute lung injury (Ali) has not been found. In this study, the role of MenSCs in the biotherapy of ALI was studied. In this study, we first verified the expression of MenSCs and found that the isolated cells were fusiform. The expression of CD29, CD4-, CD73-, CD90, CD105, CD34-CD45, CD11bC14, CD19, CD79a, CD117 and HLA-DR, is in line with the MSCs standard established by the International Society for Cell Therapy and is a member of MSCs. At the same time, we have confirmed that MenSCs has a good ability to proliferate in vitro and can undergo osteogenic adipogenic differentiation. In addition, we also found that MenSCs can express CD140b, CD194, CD221, and receptors of chemokines such as CD184 or related factors with chemokine ability, and can migrate under the stimulation of a variety of cytokines. We then explored the role of MenSCs in the treatment of ALI. In vitro, we found that MenSCs can migrate to the lung bronchial epithelial cells exposed to LPS and protect its activity. At the same time, we induced MenSCs in vitro, found that the cells formed flat epithelioid shape, and expressed pulmonary surfactant proteins such as SPBN-SPC and SPD. We used X-ray and histopathological sections to prove that MenSCs alleviates the inflammatory symptoms of the lungs in mice. The increase of lung dry / wet ratio and the expression of 尾 -catenin protein and the decrease of total protein of bronchoalveolar lavage fluid (BALF) indicated the improvement of lung permeability after MenSCs transplantation. The effect of MenSCs on inhibiting pulmonary inflammation was proved. At the same time, We also found that MenSCs could increase the expression of PCNA, reduce the expression of Caspase-3 and promote the repair of injured lung. Finally, we explored the possible mechanism of MenSCs in the treatment of acute lung injury. In mice, the expression of IL-10 and TGF- 尾 in serum and lung parenchyma increased, and the expression of pro-inflammatory factor IL-1 尾 and IL-6 decreased, suggesting that MenSCs may inhibit inflammation through immunomodulation. At the same time, we detected the expression of KGF and VE-cadherin, 尾 -catenin in lung parenchyma. It was found that MenSCs could promote the expression of adhesion protein, form adhesion junction, promote cell proliferation and repair after MenSCs transplantation. We used WB to detect the expression of PI3KP- gsk3 尾 -gsk3 尾 -gsk3 尾 -pSrc and Src protein in lung tissue. It was found that PI3KGsk3 尾 / 尾 -catenin and Src may be involved in the repair of lung. These results suggest that MenSCs may play a role in the treatment of acute lung injury induced by LPS in mice.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R563.8

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