本文選題：CD147 + shRNA��； 參考：《吉林大學(xué)》2010年博士論文

【摘要】： 糖尿病視網(wǎng)膜病變是糖尿病發(fā)展過程中常見的嚴(yán)重并發(fā)癥,是目前世界范圍內(nèi)致盲和視力受損的主要原因之一,目前對(duì)于其發(fā)病機(jī)制仍不十分清楚,治療效果亦不佳。CD147是一種分子量為50-60kD高度糖基化的單次跨膜糖蛋白,屬于免疫球蛋白超家族成員,在眼組織中尤其是視網(wǎng)膜色素上皮細(xì)胞中高表達(dá)。目前的研究證實(shí),CD147在多種疾病中發(fā)揮重要的作用,和MMPs、VEGF、MCT等多種重要的分子關(guān)系密切,因此推測(cè)CD147在糖尿病視網(wǎng)膜病變中可能發(fā)揮了重要的作用。RNA干擾是指由內(nèi)源性或外源性短雙鏈RNA誘導(dǎo)的同源mRNA的降解過程,可使基因的表達(dá)沉默。通過構(gòu)建針對(duì)目標(biāo)基因的siRNA或shRNA,導(dǎo)入細(xì)胞內(nèi)使目標(biāo)基因沉默,是研究基因和蛋白功能的重要工具。衣霉素作為N-糖鏈抑制劑多用于研究蛋白的糖基化。 本研究首先通過免疫組織化學(xué)的方法檢測(cè)了CD147在正常人眼球各組織中的表達(dá)情況。選取視網(wǎng)膜色素上皮細(xì)胞進(jìn)行體外培養(yǎng),觀察在低糖及高糖培養(yǎng)條件下RPE細(xì)胞中CD147的表達(dá)、細(xì)胞增殖和遷移能力變化以及VEGF、MMP-2、MMP-9變化。構(gòu)建CD147表達(dá)載體pBS/U6/CD147-shRNA,轉(zhuǎn)染到RPE中,檢測(cè)其對(duì)細(xì)胞CD147內(nèi)源性表達(dá)的抑制作用,篩選出抑制作用最強(qiáng)的重組質(zhì)粒,并觀察其對(duì)細(xì)胞增殖、運(yùn)動(dòng)性以及對(duì)MMP-2、MMP-9和VEGF表達(dá)的影響,證實(shí)CD147作為VEGF及MMP-9的上游因子在高糖條件下視網(wǎng)膜色素上皮細(xì)胞的生物學(xué)特性的變化中起到了重要的作用。在培養(yǎng)基中加入衣霉素,使CD147蛋白去糖基化,觀察CD147的表達(dá)變化、RPE細(xì)胞增殖和遷移能力變化以及VEGF、MMP-2、MMP-9變化,提示CD147主要是通過自身的糖基化程度不同來發(fā)揮作用,使蛋白去糖基化后可有效抑制其功能。為研究糖尿病視網(wǎng)膜病變的發(fā)病機(jī)制及治療提供新的思路。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R587.1;R774.1

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本文編號(hào)：2029658