CD147对高糖培养下RPE细胞生物学行为影响及衣霉素对CD147抑制作用研究

发布时间：2018-06-17 04:21

本文选题：CD147 + shRNA　；参考：《吉林大学》2010年博士论文

【摘要】： 糖尿病视网膜病变是糖尿病发展过程中常见的严重并发症,是目前世界范围内致盲和视力受损的主要原因之一,目前对于其发病机制仍不十分清楚,治疗效果亦不佳。CD147是一种分子量为50-60kD高度糖基化的单次跨膜糖蛋白,属于免疫球蛋白超家族成员,在眼组织中尤其是视网膜色素上皮细胞中高表达。目前的研究证实,CD147在多种疾病中发挥重要的作用,和MMPs、VEGF、MCT等多种重要的分子关系密切,因此推测CD147在糖尿病视网膜病变中可能发挥了重要的作用。RNA干扰是指由内源性或外源性短双链RNA诱导的同源mRNA的降解过程,可使基因的表达沉默。通过构建针对目标基因的siRNA或shRNA,导入细胞内使目标基因沉默,是研究基因和蛋白功能的重要工具。衣霉素作为N-糖链抑制剂多用于研究蛋白的糖基化。本研究首先通过免疫组织化学的方法检测了CD147在正常人眼球各组织中的表达情况。选取视网膜色素上皮细胞进行体外培养,观察在低糖及高糖培养条件下RPE细胞中CD147的表达、细胞增殖和迁移能力变化以及VEGF、MMP-2、MMP-9变化。构建CD147表达载体pBS/U6/CD147-shRNA,转染到RPE中,检测其对细胞CD147内源性表达的抑制作用,筛选出抑制作用最强的重组质粒,并观察其对细胞增殖、运动性以及对MMP-2、MMP-9和VEGF表达的影响,证实CD147作为VEGF及MMP-9的上游因子在高糖条件下视网膜色素上皮细胞的生物学特性的变化中起到了重要的作用。在培养基中加入衣霉素,使CD147蛋白去糖基化,观察CD147的表达变化、RPE细胞增殖和迁移能力变化以及VEGF、MMP-2、MMP-9变化,提示CD147主要是通过自身的糖基化程度不同来发挥作用,使蛋白去糖基化后可有效抑制其功能。为研究糖尿病视网膜病变的发病机制及治疗提供新的思路。
[Abstract]:Diabetic retinopathy is a common and serious complication in the development of diabetes mellitus. It is one of the main causes of blindness and visual impairment in the world at present. At present, the pathogenesis of diabetic retinopathy is still unclear. CD147 is a single transmembrane glycoprotein with a molecular weight of 50-60 KD, which belongs to the immunoglobulin superfamily and is highly expressed in eye tissues, especially in retinal pigment epithelial cells. Current studies have confirmed that CD147 plays an important role in many diseases and is closely related to many important molecules such as MMPsVEGFCT. It is speculated that CD147 may play an important role in diabetic retinopathy. RNA interference refers to the degradation of homologous mRNA induced by endogenous or exogenous short double-stranded RNA, which silences gene expression. It is an important tool to study the function of gene and protein by constructing siRNA or shRNA targeting gene and silencing target gene into cells. As an inhibitor of N-sugar chain, itamycin is used to study the glycosylation of protein. In this study, the expression of CD147 in normal human eye tissues was detected by immunohistochemical method. RPE cells were cultured in vitro to observe the expression of CD147, the changes of cell proliferation and migration ability and the changes of VEGF MMP-2 MMP-9 in RPE cells cultured with low glucose or high glucose. CD147 expression vector pBS- / U6 / CD147-shRNA was constructed and transfected into RPE to detect its inhibitory effect on the endogenous expression of CD147, to screen the recombinant plasmid with the strongest inhibitory effect, and to observe its effects on cell proliferation, motility and the expression of MMP-9 and VEGF. CD147, as the upstream factor of VEGF and MMP-9, plays an important role in the changes of the biological characteristics of retinal pigment epithelial cells under high glucose condition. The CD147 protein was deglycosylated by adding chlortetracycline to the culture medium. The changes of CD147 expression and the proliferation and migration ability of RPE cells, as well as the changes of VEGFU MMP-2MMP-9, suggested that CD147 acted mainly through its own glycosylation degree. The deglycosylation of protein can effectively inhibit its function. To provide a new way to study the pathogenesis and treatment of diabetic retinopathy.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R587.1;R774.1

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