本文選題：AQP5　切入點：骨髓起源間充質(zhì)干細(xì)胞　出處：《東北師范大學(xué)》2012年博士論文

【摘要】：水通道蛋白（Aquaporins，AQPs）是一類廣泛表達(dá)在原核和真核細(xì)胞膜上快速轉(zhuǎn)運水分子的特異性孔道。哺乳動物水通道蛋白家族包括13個成員（AQP0-AQP12），它們在機體內(nèi)選擇性地表達(dá)于多種組織和器官，并行使重要的生理功能。除了完成各種上皮和內(nèi)皮組織的液體轉(zhuǎn)運外，在其它生命過程中也行使重要的功能，包括細(xì)胞遷移、增殖、凋亡、細(xì)胞體積調(diào)控、神經(jīng)信號轉(zhuǎn)導(dǎo)、線粒體新陳代謝、免疫細(xì)胞功能等，，從而影響機體血管生成、腫瘤細(xì)胞的生長與侵入、組織損傷修復(fù)、維持細(xì)胞穩(wěn)態(tài)和免疫系統(tǒng)功能等多種重要的生命活動。近些年來，人們對水通道蛋白結(jié)構(gòu)與功能的研究已經(jīng)廣泛的開展，所使用的研究方法與手段也比較豐富，關(guān)于水通道蛋白生理功能的研究不斷有新的重要發(fā)現(xiàn)。但是，水通道蛋白在干細(xì)胞功能中的作用研究很少。有報道稱水通道蛋白在神經(jīng)干細(xì)胞上表達(dá)，并影響其增殖、遷移和神經(jīng)分化，這些結(jié)果提示我們水通道蛋白可能在間充質(zhì)干細(xì)胞的生理功能中發(fā)揮重要的作用。 間充質(zhì)干細(xì)胞（Mesenchymal stem cells，MSCs）是一群異質(zhì)類具有自我更新能力和多向分化潛能的成體干細(xì)胞，廣泛分布于多種組織和器官。由于MSCs來源豐富、體外增殖能力強、多項分化潛能、免疫原性低等優(yōu)點，在以細(xì)胞為基礎(chǔ)的組織損傷修復(fù)診療中一直是研究的熱點。本研究中首先通過RT-PCR、Western Blot和免疫熒光等分子生物學(xué)手段發(fā)現(xiàn)水通道蛋白AQP5在骨髓起源的間充質(zhì)干細(xì)胞（bone marrow-derivedMSCs，BMSCs）質(zhì)膜高表達(dá)，而后發(fā)現(xiàn)AQP5敲除小鼠BMSCs的質(zhì)膜水通透性顯著低于野生鼠。并通過免疫熒光、克隆形成實驗和細(xì)胞計數(shù)實驗發(fā)現(xiàn)兩種基因型BMSCs在細(xì)胞形態(tài)和增殖能力方面沒有顯著差異。但是在多項分化潛能方面，通過定向誘導(dǎo)后用油紅O染色（成脂肪），茜素紅S和堿性磷酸酶染色（成骨），Collagen II和番紅素O染色（成軟骨）發(fā)現(xiàn)AQP5敲除顯著提高了BMSCs的成脂、成骨和成軟骨分化潛能。運用尼羅紅定量、鈣離子、堿性磷酸酶和胞外糖胺多糖含量的測定和通過檢測三系誘導(dǎo)分化特異標(biāo)記分子（PPARγ2, C/EBPα, adipsin, collagen1a, osteopontin,ALP, collagen11a,collagen2a和aggrecan）的相對mRNA表達(dá)水平進(jìn)一步證實了上述結(jié)果。在鉆孔骨損傷修復(fù)實驗中發(fā)現(xiàn)，損傷后14和21天，AQP5基因敲除小鼠骨損傷修復(fù)速度顯著快于野生鼠。在對這一表型的機理研究中，我們發(fā)現(xiàn)AQP5敲除明顯降低了分化中BMSCs的凋亡率。應(yīng)用凋亡抑制劑Z-VAD-FMK結(jié)果顯示Z-VAD-FMK顯著提高了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。這一結(jié)果表明AQP5介導(dǎo)的高質(zhì)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。本研究首次發(fā)現(xiàn)AQP5在BMSCs質(zhì)膜上表達(dá)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可能成為調(diào)控組織再生和損傷修復(fù)的新分子靶標(biāo)。
[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- 緯 2, C/EBP 偽, 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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 焦光宇,李爾然,于潤江;內(nèi)毒素誘導(dǎo)的急性肺損傷大鼠呼吸膜AQP1及AQP5表達(dá)的研究(英文)[J];Chinese Medical Journal;2002年07期

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