转甲状腺素蛋白在糖尿病视网膜病变新生血管生成中的阻遏作用及机制研究

发布时间：2018-05-06 01:25

本文选题：转甲状腺素蛋白 + 糖尿病视网膜　；参考：《南京医科大学》2017年博士论文

【摘要】：目的:转甲状腺素蛋白(TTR)可诱导人脐静脉细胞的凋亡。在人眼组织中,转甲状腺素蛋白主要被视网膜色素上皮(RPE)细胞分泌。而TTR影响糖尿病视网膜新生血管的形成尚未阐明。本文旨在模拟糖尿病视网膜病变眼部高糖及缺氧条件,探讨TTR对高葡萄糖和缺氧环境下视网膜新生血管形成的生成作用和机制。方法:正常,高葡萄糖和缺氧的环境下培养人类视网膜微血管内皮细胞(HRMECs)。正常血糖浓度约为5.5mmol/L,25mmol/L葡萄糖模拟高血糖,而用2000μmol/L CoCl 2诱导缺氧环境。并在正常和高糖及缺氧的培养基中培养4μmol/L转甲状腺素蛋白。通过模拟不同环境,观察TTR对HRMECs和人视网膜色素上皮细胞(hRPEC)增殖的影响。进一步研究TTR对HRMECs的生物行为学的影响,同样是在体外模拟正常和病理环境下,并在共培养环境模拟体内环境,通过TTR的过量表达,体外愈伤实验和血管形成测定等实验进行验证细胞生物行为学的改变。在初步探讨机制方面,通过实时荧光定量PCR(qRT-PCR)研究糖尿病视网膜血管生长因子相关基因如Tie2,VEGFR1,VEGFR2,Angpt1和Angpt2 的 mRNA 水平。结果:在模拟体内高糖环境及缺氧环境下,我们观察到HRMECs和hRPEC的增殖是降低的。此外,在高糖环境下,外源性的添加4μmol/LTTR可明显抑制内皮细胞的增殖。利用愈伤和成管实验,在正常和病理条件共培养环境下同样证明,模拟高血糖和缺氧的糖尿病视网膜病变环境下,hRPECs均能显著抑制HRMECs的增殖。这些结果均表明,无论加入外源或内源性TTR,仅在高血糖条件下,TTR可抑制内皮细胞的生成。此外,在转录水平的研究中,外源性的加入TTR在高血糖和高血糖/缺氧环境中,Tie2和Angpt1 mRNA水平上升,而VEGFR1,VEGFR2和Angpt1的mRNA水平被抑制。结论:高葡萄糖和缺氧模拟的病理环境下,特别是高糖环境中外源性添加TTR可抑制HRMECs生长。同样HRPE分泌的TTR在相同的环境中也同样可抑制HRMECs的生长。而新生血管形成的关键基因包括Tie2,VEGFR1,VEGFR2,Angpt1和Angpt2受TTR调控。在DR条件下,TTR通过抑制HRMECs的增殖,迁移和管形成来显着抑制新生血管形成。
[Abstract]:Objective: TTRT can induce apoptosis of human umbilical vein cells. In human eye tissue, Thyroxine protein is mainly secreted by retinal pigment epithelium (RPE) cells. However, the effect of TTR on retinal neovascularization in diabetic patients has not been clarified. The aim of this study was to investigate the role and mechanism of TTR in retinal neovascularization in hyperglycemic and hypoxic environments by simulating the ocular hyperglycemia and hypoxia conditions of diabetic retinopathy. Methods: human retinal microvascular endothelial cells (HRMECs) were cultured in normal, hyperglycemic and anoxic environment. The normal glucose concentration was about 5.5 mmol / L and 25 mmol / L glucose simulated hyperglycemia, while 2000 渭 mol/L CoCl _ 2 was used to induce anoxic environment. 4 渭 mol/L Thyroxine protein was cultured in normal medium, high glucose medium and hypoxia medium. The effects of TTR on the proliferation of HRMECs and human retinal pigment epithelial cells (RPE) were observed by simulating different environments. To further study the effect of TTR on the biological behavior of HRMECs, we also simulated in vitro normal and pathological environment, and in co-culture environment, through the overexpression of TTR. In vitro callus assay and angiogenesis assay were used to verify the changes of cellular biological behavior. In order to explore the mechanism, the mRNA levels of vascular growth factor related genes such as Tie2VEGFR1VEGFR2VEGFR2 Angpt1 and Angpt2 were studied by real-time fluorescence quantitative PCRQRT-PCR. Results: we observed that the proliferation of HRMECs and hRPEC was decreased in simulated hyperglycemic and anoxic environment. In addition, exogenous addition of 4 渭 mol/LTTR significantly inhibited the proliferation of endothelial cells in high glucose environment. Using callus and tube formation experiments, it was also proved that hRPECs could significantly inhibit the proliferation of HRMECs in diabetic retinopathy with hypoglycemia and hypoxia under normal and pathological conditions. These results indicated that TTRR could inhibit endothelial cell formation only under hyperglycemia. In addition, exogenous addition of TTR increased the levels of Tie2 and Angpt1 mRNA in hyperglycemia and hyperglycemia / hypoxia, while VEGFR1VEGFR2 and Angpt1 mRNA levels were inhibited. Conclusion: exogenous addition of TTR can inhibit the growth of HRMECs in high glucose and anoxic simulated pathological environment, especially in high glucose environment. TTR secreted by the same HRPE also inhibited the growth of HRMECs in the same environment. The key genes of neovascularization include Tie2VEGFR1, VEGFR2Angpt1 and Angpt2 regulated by TTR. In Dr condition TTR significantly inhibited angiogenesis by inhibiting proliferation migration and tube formation of HRMECs.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R774.1

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