發(fā)布時(shí)間：2018-05-06 01:25

  本文選題：轉(zhuǎn)甲狀腺素蛋白 + 糖尿病視網(wǎng)膜　； 參考：《南京醫(yī)科大學(xué)》2017年博士論文

【摘要】：目的:轉(zhuǎn)甲狀腺素蛋白(TTR)可誘導(dǎo)人臍靜脈細(xì)胞的凋亡。在人眼組織中,轉(zhuǎn)甲狀腺素蛋白主要被視網(wǎng)膜色素上皮(RPE)細(xì)胞分泌。而TTR影響糖尿病視網(wǎng)膜新生血管的形成尚未闡明。本文旨在模擬糖尿病視網(wǎng)膜病變眼部高糖及缺氧條件,探討TTR對高葡萄糖和缺氧環(huán)境下視網(wǎng)膜新生血管形成的生成作用和機(jī)制。方法:正常,高葡萄糖和缺氧的環(huán)境下培養(yǎng)人類視網(wǎng)膜微血管內(nèi)皮細(xì)胞(HRMECs)。正常血糖濃度約為5.5mmol/L,25mmol/L葡萄糖模擬高血糖,而用2000μmol/L CoCl 2誘導(dǎo)缺氧環(huán)境。并在正常和高糖及缺氧的培養(yǎng)基中培養(yǎng)4μmol/L轉(zhuǎn)甲狀腺素蛋白。通過模擬不同環(huán)境,觀察TTR對HRMECs和人視網(wǎng)膜色素上皮細(xì)胞(hRPEC)增殖的影響。進(jìn)一步研究TTR對HRMECs的生物行為學(xué)的影響,同樣是在體外模擬正常和病理環(huán)境下,并在共培養(yǎng)環(huán)境模擬體內(nèi)環(huán)境,通過TTR的過量表達(dá),體外愈傷實(shí)驗(yàn)和血管形成測定等實(shí)驗(yàn)進(jìn)行驗(yàn)證細(xì)胞生物行為學(xué)的改變。在初步探討機(jī)制方面,通過實(shí)時(shí)熒光定量PCR(qRT-PCR)研究糖尿病視網(wǎng)膜血管生長因子相關(guān)基因如Tie2,VEGFR1,VEGFR2,Angpt1和Angpt2 的 mRNA 水平。結(jié)果:在模擬體內(nèi)高糖環(huán)境及缺氧環(huán)境下,我們觀察到HRMECs和hRPEC的增殖是降低的。此外,在高糖環(huán)境下,外源性的添加4μmol/LTTR可明顯抑制內(nèi)皮細(xì)胞的增殖。利用愈傷和成管實(shí)驗(yàn),在正常和病理?xiàng)l件共培養(yǎng)環(huán)境下同樣證明,模擬高血糖和缺氧的糖尿病視網(wǎng)膜病變環(huán)境下,hRPECs均能顯著抑制HRMECs的增殖。這些結(jié)果均表明,無論加入外源或內(nèi)源性TTR,僅在高血糖條件下,TTR可抑制內(nèi)皮細(xì)胞的生成。此外,在轉(zhuǎn)錄水平的研究中,外源性的加入TTR在高血糖和高血糖/缺氧環(huán)境中,Tie2和Angpt1 mRNA水平上升,而VEGFR1,VEGFR2和Angpt1的mRNA水平被抑制。結(jié)論:高葡萄糖和缺氧模擬的病理環(huán)境下,特別是高糖環(huán)境中外源性添加TTR可抑制HRMECs生長。同樣HRPE分泌的TTR在相同的環(huán)境中也同樣可抑制HRMECs的生長。而新生血管形成的關(guān)鍵基因包括Tie2,VEGFR1,VEGFR2,Angpt1和Angpt2受TTR調(diào)控。在DR條件下,TTR通過抑制HRMECs的增殖,遷移和管形成來顯著抑制新生血管形成。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R774.1

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本文編號：1850165