水通道蛋白AQP5在小鼠骨髓间充质干细胞分化中的功能研究

发布时间：2018-04-02 06:52

本文选题：AQP5　切入点：骨髓起源间充质干细胞　出处：《东北师范大学》2012年博士论文

【摘要】：水通道蛋白（Aquaporins，AQPs）是一类广泛表达在原核和真核细胞膜上快速转运水分子的特异性孔道。哺乳动物水通道蛋白家族包括13个成员（AQP0-AQP12），它们在机体内选择性地表达于多种组织和器官，并行使重要的生理功能。除了完成各种上皮和内皮组织的液体转运外，在其它生命过程中也行使重要的功能，包括细胞迁移、增殖、凋亡、细胞体积调控、神经信号转导、线粒体新陈代谢、免疫细胞功能等，，从而影响机体血管生成、肿瘤细胞的生长与侵入、组织损伤修复、维持细胞稳态和免疫系统功能等多种重要的生命活动。近些年来，人们对水通道蛋白结构与功能的研究已经广泛的开展，所使用的研究方法与手段也比较丰富，关于水通道蛋白生理功能的研究不断有新的重要发现。但是，水通道蛋白在干细胞功能中的作用研究很少。有报道称水通道蛋白在神经干细胞上表达，并影响其增殖、迁移和神经分化，这些结果提示我们水通道蛋白可能在间充质干细胞的生理功能中发挥重要的作用。间充质干细胞（Mesenchymal stem cells，MSCs）是一群异质类具有自我更新能力和多向分化潜能的成体干细胞，广泛分布于多种组织和器官。由于MSCs来源丰富、体外增殖能力强、多项分化潜能、免疫原性低等优点，在以细胞为基础的组织损伤修复诊疗中一直是研究的热点。本研究中首先通过RT-PCR、Western Blot和免疫荧光等分子生物学手段发现水通道蛋白AQP5在骨髓起源的间充质干细胞（bone marrow-derivedMSCs，BMSCs）质膜高表达，而后发现AQP5敲除小鼠BMSCs的质膜水通透性显著低于野生鼠。并通过免疫荧光、克隆形成实验和细胞计数实验发现两种基因型BMSCs在细胞形态和增殖能力方面没有显著差异。但是在多项分化潜能方面，通过定向诱导后用油红O染色（成脂肪），茜素红S和碱性磷酸酶染色（成骨），Collagen II和番红素O染色（成软骨）发现AQP5敲除显著提高了BMSCs的成脂、成骨和成软骨分化潜能。运用尼罗红定量、钙离子、碱性磷酸酶和胞外糖胺多糖含量的测定和通过检测三系诱导分化特异标记分子（PPARγ2, C/EBPα, adipsin, collagen1a, osteopontin,ALP, collagen11a,collagen2a和aggrecan）的相对mRNA表达水平进一步证实了上述结果。在钻孔骨损伤修复实验中发现，损伤后14和21天，AQP5基因敲除小鼠骨损伤修复速度显著快于野生鼠。在对这一表型的机理研究中，我们发现AQP5敲除明显降低了分化中BMSCs的凋亡率。应用凋亡抑制剂Z-VAD-FMK结果显示Z-VAD-FMK显著提高了AQP5敲除小鼠和野生鼠BMSCs的分化能力，但野生鼠提高的幅度更大。这一结果表明AQP5介导的高质膜水通透性提高了分化中BMSCs的凋亡率，因而降低了其分化能力。本研究首次发现AQP5在BMSCs质膜上表达并且在BMSCs分化能力方面可能具有重要作用，提示水通道蛋白AQP5可能成为调控组织再生和损伤修复的新分子靶标。
[Abstract]:Aquaporins aquaporins (aquaporins) is a kind of special pore which is widely expressed in prokaryotic and eukaryotic cell membrane to transport water molecules rapidly.The mammalian aquaporin family consists of 13 members, AQP0-AQP12, which are selectively expressed in a variety of tissues and organs and perform important physiological functions.In addition to completing the liquid transport of various epithelial and endothelial tissues, it also performs important functions in other life processes, including cell migration, proliferation, apoptosis, cell volume regulation, neural signal transduction, mitochondrial metabolism,The function of immune cells affects many important life activities, such as angiogenesis, tumor cell growth and invasion, tissue damage and repair, maintenance of cell homeostasis and immune system function.In recent years, the research on the structure and function of aquaporins has been widely carried out, and the research methods and methods used are also relatively rich. The research on the physiological functions of aquaporins has made new and important discoveries.However, the role of aquaporins in stem cell function is rarely studied.It has been reported that aquaporins are expressed on neural stem cells and affect their proliferation, migration and neural differentiation. These results suggest that aquaporins may play an important role in the physiological function of mesenchymal stem cells.Mesenchymal stem cells (MSCs) are a group of heterogeneous adult stem cells with self-renewal and multi-differentiation potential, which are widely distributed in various tissues and organs.Because of the rich sources of MSCs, strong proliferation in vitro, multiple differentiation potential, low immunogenicity and other advantages, it has been a hot spot in the diagnosis and treatment of cell-based tissue injury repair.In this study, we first found the high expression of aquaporin AQP5 in the plasma membrane of bone marrow derived mesenchymal stem cells (MSCs) by RT-PCR- Western Blot and immunofluorescence, but then found that the membrane water permeability of BMSCs in AQP5 knockout mice was significantly lower than that in wild mice.The results of immunofluorescence, clone formation and cell counting showed that there was no significant difference between the two genotypes BMSCs in cell morphology and proliferation ability.However, in terms of differentiation potential, AQP5 knockout significantly increased the fat-forming of BMSCs through directed induction with oil red O staining (adipose formation, alizarin red S and alkaline phosphatase staining (osteoblast collagen II and guanosine O staining).Osteogenic and chondrogenic potential.The above results were further confirmed by the quantitative determination of Nile red, calcium ion, alkaline phosphatase and extracellular glycosaminoglycan polysaccharides, and the relative mRNA expression levels of PPAR- 纬 2, C/EBP 伪, adipsin, collagen1a, osteopontinin ALPa, collagen11a collagen2a and aggrecan.In the experiment of repairing bone injury with borehole, it was found that the repair speed of bone injury in mice with AQP5 gene knockout was significantly faster than that in wild mice at 14 and 21 days after injury.In the study of the mechanism of this phenotype, we found that AQP5 knockout significantly reduced the apoptosis rate of BMSCs in differentiation.The results of apoptosis inhibitor Z-VAD-FMK showed that Z-VAD-FMK significantly increased the differentiation ability of BMSCs in AQP5 knockout mice and wild mice, but in wild mice, the increase was more significant.The results suggested that high membrane water permeability mediated by AQP5 increased the apoptosis rate of BMSCs in differentiation and reduced its differentiation ability.It is the first time that AQP5 is expressed on the plasma membrane of BMSCs and may play an important role in the differentiation of BMSCs, suggesting that aquaporin AQP5 may be a new molecular target for regulating tissue regeneration and damage repair.
【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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