本文關(guān)鍵詞： microRNA-483-3p 小梁網(wǎng)細(xì)胞 Smad4 細(xì)胞外基質(zhì) 青光眼　出處：《天津醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：背景與目的:青光眼是世界第二位致盲眼病,是由于病理性高眼壓導(dǎo)致視神經(jīng)損傷而出現(xiàn)特征性的視野損害,其主要因素是眼內(nèi)壓(intraocular pressure,IOP)升高。目前認(rèn)為小梁網(wǎng)細(xì)胞(trabecular meshwork,TMC)的細(xì)胞外基質(zhì)(extracellular matrix,ECM)過(guò)度沉積,使房水(aqueous humor,AH)流出阻力增加,從而使眼內(nèi)壓升高。本項(xiàng)研究主要探究了在氧化應(yīng)激條件下,micro RNA-483-3p(mi R-483-3p)對(duì)小梁網(wǎng)細(xì)胞ECM的影響,并明確了mi R-483-3p調(diào)節(jié)ECM產(chǎn)生的機(jī)制。方法:首先我們利用Western blot技術(shù)檢測(cè)了小梁網(wǎng)細(xì)胞在氧化應(yīng)激狀態(tài)下ECM成分(fibronectin,laminin,collagen I)的表達(dá)水平,并利用實(shí)時(shí)定量PCR(q PCR)檢測(cè)了小梁網(wǎng)細(xì)胞中mi R-483-3p的表達(dá)水平。此后,我們利用慢病毒載體穩(wěn)定表達(dá)pri-mi R-483,并分別用Western blot與q PCR檢測(cè)mi R-483-3p對(duì)ECM的調(diào)節(jié)作用。此外,我們通過(guò)m RNA靶點(diǎn)預(yù)測(cè)軟件尋找候選靶點(diǎn),并用雙熒光素酶檢測(cè)及Western blot確定mi R-483-3p最終靶點(diǎn)為Smad4。最后,我們?cè)谛×壕W(wǎng)細(xì)胞中敲低Smad4基因,再次用Western blot與q PCR檢測(cè)ECM表達(dá)水平。結(jié)果:在氧化應(yīng)激條件下,小梁網(wǎng)細(xì)胞ECM成分(fibronectin,laminin,collagen I)的表達(dá)水平在m RNA和蛋白水平均有明顯上升;而mi R-483-3p的表達(dá)水平下降。對(duì)小梁網(wǎng)細(xì)胞進(jìn)行外源性表達(dá)mi R-483-3p,可以導(dǎo)致其ECM表達(dá)水平下降。此外,mi R-483-3p通過(guò)兩個(gè)結(jié)合部位與Smad4直接靶定,從而導(dǎo)致Smad4蛋白表達(dá)水平下降。最后,在小梁網(wǎng)細(xì)胞中敲低Smad4后,我們觀察到ECM表達(dá)在m RNA和蛋白水平均有下調(diào)。結(jié)論:在小梁網(wǎng)細(xì)胞中,mi R-483-3p通過(guò)下調(diào)Smad4蛋白表達(dá),可以抑制ECM成分的產(chǎn)生。這種有效的抑制作用,可能存在降低眼內(nèi)壓的效果,這表明mi R-483-3p可能是青光眼治療的潛在新靶點(diǎn)。
[Abstract]:Background & objective: glaucoma is the second leading cause of blindness in the world, which is characterized by visual field damage due to pathological high intraocular pressure (IOP). The main factor is the increase of intraocular pressure (IOP). Trabecular meshwork is currently considered as trabecular meshwork network cells. The extracellular matrix (extracellular matrix ECM) overdeposition of TMC increased the outflow resistance of aqueous humor humorus (AH). In this study, we investigated the effect of micro RNA-483-3p(mi R-483-3p on ECM in trabecular meshwork cells under oxidative stress. The mechanism by which mi R-483-3p regulates the production of ECM is clarified. Methods: first, we use Western. Blot technique was used to detect the components of ECM in trabecular meshwork cells under oxidative stress. Fibronectin. The expression level of laminin collagen I. The expression of miR-483-3p in trabecular meshwork cells was detected by real-time quantitative PCR(q. We expressed pri-mi R-483 stably using lentivirus vector. Western blot and Q PCR were used to detect the regulatory effect of mi R-483-3p on ECM. In addition, we used m RNA target prediction software to find candidate targets. The final target of mi R-483-3p was identified as Smad4by double luciferase detection and Western blot. Finally, we knocked down the Smad4 gene in trabecular meshwork cells. Western blot and Q PCR were used to detect the expression of ECM. Results: under oxidative stress, the ECM component of trabecular meshwork cells was fibronectin. The expression level of laminine collagen I was significantly increased at the level of m RNA and protein. However, the expression level of mi R-483-3p decreased. Exogenous expression of mi R-483-3p in trabecular meshwork cells resulted in the decrease of ECM expression level. Mi R-483-3p directly targeted Smad4 through two binding sites, resulting in a decrease in Smad4 protein expression. Finally, Smad4 was knocked down in trabecular meshwork cells. We observed that the expression of ECM was down-regulated at the level of m RNA and protein. Conclusion: in trabecular meshwork cells, ECM R-483-3p down-regulates the expression of Smad4 protein. This effective inhibition may have the effect of lowering intraocular pressure, which suggests that mi R-483-3p may be a potential new target for glaucoma therapy.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R775

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