miR-483-3p调节人小梁网细胞的细胞外基质表达及其机制研究

发布时间：2018-01-30 16:08

本文关键词： microRNA-483-3p 小梁网细胞 Smad4 细胞外基质青光眼　出处：《天津医科大学》2016年博士论文　论文类型：学位论文

【摘要】：背景与目的:青光眼是世界第二位致盲眼病,是由于病理性高眼压导致视神经损伤而出现特征性的视野损害,其主要因素是眼内压(intraocular pressure,IOP)升高。目前认为小梁网细胞(trabecular meshwork,TMC)的细胞外基质(extracellular matrix,ECM)过度沉积,使房水(aqueous humor,AH)流出阻力增加,从而使眼内压升高。本项研究主要探究了在氧化应激条件下,micro RNA-483-3p(mi R-483-3p)对小梁网细胞ECM的影响,并明确了mi R-483-3p调节ECM产生的机制。方法:首先我们利用Western blot技术检测了小梁网细胞在氧化应激状态下ECM成分(fibronectin,laminin,collagen I)的表达水平,并利用实时定量PCR(q PCR)检测了小梁网细胞中mi R-483-3p的表达水平。此后,我们利用慢病毒载体稳定表达pri-mi R-483,并分别用Western blot与q PCR检测mi R-483-3p对ECM的调节作用。此外,我们通过m RNA靶点预测软件寻找候选靶点,并用双荧光素酶检测及Western blot确定mi R-483-3p最终靶点为Smad4。最后,我们在小梁网细胞中敲低Smad4基因,再次用Western blot与q PCR检测ECM表达水平。结果:在氧化应激条件下,小梁网细胞ECM成分(fibronectin,laminin,collagen I)的表达水平在m RNA和蛋白水平均有明显上升;而mi R-483-3p的表达水平下降。对小梁网细胞进行外源性表达mi R-483-3p,可以导致其ECM表达水平下降。此外,mi R-483-3p通过两个结合部位与Smad4直接靶定,从而导致Smad4蛋白表达水平下降。最后,在小梁网细胞中敲低Smad4后,我们观察到ECM表达在m RNA和蛋白水平均有下调。结论:在小梁网细胞中,mi R-483-3p通过下调Smad4蛋白表达,可以抑制ECM成分的产生。这种有效的抑制作用,可能存在降低眼内压的效果,这表明mi R-483-3p可能是青光眼治疗的潜在新靶点。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R775

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