本文選題：人視網(wǎng)膜色素上皮細胞 + 人臍靜脈內(nèi)皮細胞��； 參考：《昆明醫(yī)科大學》2016年博士論文

【摘要】：目的通過體外實驗研究探討非諾貝特對人視網(wǎng)膜色素上皮細胞(RPE)和人臍靜脈內(nèi)皮細胞(HUVEC)活力的影響及可能的機制,通過體內(nèi)實驗探討激光誘導BN大鼠脈絡膜新生血管形成過程中脈絡膜和視網(wǎng)膜內(nèi)VEGFC和VEGFR-3的表達變化規(guī)律及非諾貝特對脈絡膜新生血管發(fā)生發(fā)展的影響作用,為眼脈絡膜新生血管性相關疾病提供新的思路。方法1.將RPE和HUVEC細胞分組：RPE正常組,RPE+非諾貝特組,RPE缺氧組,RPE缺氧+非諾貝特組；HUVEC細胞正常組,]HUVEC+缺氧上清組；將健康成年BN大鼠隨機分成空白對照組和實驗組,實驗組分為非諾貝特干預組(用藥組),非干預組(實驗對照組),每組以1w、2w、3w和4w為觀察點。2.用超氧化物陰離子探針檢測RPE缺氧情況,MTT檢測細胞活力,ELISA檢測RPE細胞培養(yǎng)液上清中VEGFC和VEGFR-3的表達；細胞劃痕實驗檢測細胞遷移能力,細胞管腔形成實驗檢測]HUVEC細胞的血管形成能力,qRT-PCR和Western-blot檢測RPE細胞VEGFC和VEGFR-3的mRNA和蛋白質(zhì)的表達水平；用FFA檢測非諾貝特干預組和非干預組激光斑處不同時間點脈絡膜新生血管的滲漏面積和滲漏強度,用凝聚素B4染脈絡膜鋪片觀察激光斑處不同時間點新生血管內(nèi)皮細胞的染色程度,行BN大鼠眼球冰凍切片,用免疫熒光化學檢測激光斑處脈絡膜和視網(wǎng)膜VEGFC和VEGFR-3的分布情況,用qRT-PCR技術定量檢測不同時間點各組脈絡膜和視網(wǎng)膜內(nèi)VEGFC和VEGFR-3的mRNA表達變化,用Western Blot技術,檢測不同時間點各組脈絡膜和視網(wǎng)膜內(nèi)VEGFC和VEGFR-3的蛋白質(zhì)表達變化。結果1.非諾貝特能抑制因缺氧引起的RPE細胞內(nèi)和培養(yǎng)液上清中VEGFC和VEGFR-3表達上調(diào)；2.缺氧培養(yǎng)RPE細胞上清液可促進HUVEC細胞活力增加,細胞遷移數(shù)增多,管腔形成數(shù)增多,而非諾貝特能抑制這一過程；3.激光誘導BN大鼠脈絡膜新生血管形成后,脈絡膜和視網(wǎng)膜內(nèi)均可以發(fā)現(xiàn)有VEGFC和VEGFR-3的表達,其中VEGFC主要表達于激光斑上方視網(wǎng)膜神經(jīng)節(jié)細胞層內(nèi),VEGFR-3主要表達與脈絡膜激光斑處的新生血管區(qū)域,正常的脈絡膜和視網(wǎng)膜未發(fā)現(xiàn)有上訴兩種因子的表達；4.激光誘導BN大鼠脈絡膜新生血管形成后,脈絡膜和視網(wǎng)膜表達的VEGFC和VEGFR-3呈現(xiàn)一定的規(guī)律性,在脈絡膜和視網(wǎng)膜中VEGFC表達隨時間的推移呈現(xiàn)逐漸下調(diào)的趨勢,在脈絡膜中VEGFR-3的表達呈現(xiàn)逐漸升高并持續(xù)維持于高表達水平,在視網(wǎng)膜中VEGFR-3基本不表達或僅有少量表達；5.非諾貝特能夠明顯下調(diào)激光誘導BN大鼠脈絡膜新生血管形成和發(fā)展過程,其能夠明顯下調(diào)視網(wǎng)膜和脈絡膜內(nèi)VEGFC的表達量,脈絡膜內(nèi)的VEGFR-3的表達下調(diào)與非諾貝特下調(diào)VEGFC的表達量有關。結論1.CoCl2誘導體外培養(yǎng)的RPE細胞缺氧導致VEGFC和VEGFR-3表達上調(diào),非諾貝特能夠抑制RPE細胞缺氧狀態(tài)下VEGFC和VEGFR-3的表達上調(diào)；2.RPE缺氧后的細胞培養(yǎng)上清液中的VEGFC和VEGFR-3濃度上升,上清液對HUVEC細胞具有促增殖、遷移和毛細血管成管活性的作用,提示RPE細胞在缺氧導致的CNV形成過程中起到了重要的作用,非諾貝特能夠抑制RPE細胞缺氧后的VEGFC和VEGFR-3的上調(diào),從而有可能通過調(diào)節(jié)RPE的功能活性來間接影響HUVEC細胞的活性從而達到抑制新生血管的形成。3.VEGFC主要表達于BN大鼠激光損傷區(qū)域上方的視網(wǎng)膜神經(jīng)節(jié)細胞層內(nèi),VEGFR-3主要表達于激光斑處的脈絡膜損傷區(qū)；4.非諾貝特能夠下調(diào)脈絡膜和視網(wǎng)膜內(nèi)VEGFC的表達,對脈絡膜內(nèi)VEGFR-3的下調(diào)作用可能是通過VEGFC的間接影響。
[Abstract]:Objective to investigate the effects and possible mechanisms of fenofibrate on human retinal pigment epithelial cells (RPE) and human umbilical vein endothelial cells (HUVEC) in vitro, and to explore the changes in the expression of VEGFC and VEGFR-3 in choroidal and retina during the formation of choroidal neovascularization in BN rats by in vivo experiments. The effects of fenofibrate on the development of choroidal neovascularization, provide new ideas for the choroidal neovascularization related diseases. Methods 1. RPE and HUVEC cells were divided into RPE normal group, RPE+ fenofibrate group, RPE anoxic group, RPE anoxic + fenofibrate group, normal HUVEC cell group,]HUVEC+ hypoxia supernatant group, and healthy adult BN Rats were randomly divided into blank control group and experimental group. The experimental group was divided into Fenofibrate Intervention group (drug group), non intervention group (experimental control group), 1W, 2W, 3W and 4W were used as observation points for the detection of RPE anoxia with superoxide anion probe.2., MTT detection of cell viability, ELISA detected VEGFC and VEGFR-3 expression in the supernatant of RPE cell culture liquid. The cell migration ability was detected by cell scratch test, the angiogenesis ability of]HUVEC cells was detected by cell lumen formation, and the expression level of mRNA and protein of VEGFC and VEGFR-3 in RPE cells was detected by qRT-PCR and Western-blot, and the choroidal neovascularization at different time points at different spots in the fenofibrate and non intervention groups was detected with FFA. The coloring degree of the neovascular endothelial cells at different time points at the laser spot was observed with the leaking area and the leakage intensity. The frozen section of the eye of the BN rat was frozen. The distribution of VEGFC and VEGFR-3 in the choroid and retina at the laser spot was detected by immunofluorescence chemistry, and the different time points were measured by qRT-PCR technique. The changes in mRNA expression of VEGFC and VEGFR-3 in choroid and retina. Western Blot technique was used to detect the changes in the protein expression of VEGFC and VEGFR-3 in the choroid and retina at different time points. Results 1. fenofibrate could inhibit the up-regulated expression of VEGFC and VEGFR-3 in the RPE cells and the culture liquid supernatant caused by hypoxia; 2. anoxic culture. The supernatant of RPE cells could increase the vitality of HUVEC cells, increase the number of cell migration, increase the number of cavity formation, and fenofibrate can inhibit this process. 3. after the formation of choroidal neovascularization in BN rats, the expression of VEGFC and VEGFR-3 can be found in the choroid and retina, and VEGFC is mainly expressed above the laser spot. In the retinal ganglion cell layer, VEGFR-3 is mainly expressed in the neovascular region of the choroidal laser spot, and the expression of two factors is not found in the normal choroid and retina. 4. after the formation of the choroidal neovascularization in the BN rats, the VEGFC and VEGFR-3 in the choroid and retina present certain regularity, in the vein. The expression of VEGFC in the retinal membrane and retina gradually decreased with time. The expression of VEGFR-3 in the choroid gradually increased and maintained at the high expression level. In the retina, VEGFR-3 was basically not expressed or only a small amount of expression was expressed. 5. fenofibrate can obviously lower the laser induced choroidal neovascularization in BN rats. It can obviously reduce the expression of VEGFC in the retina and choroid, and the downregulation of VEGFR-3 in the choroid is related to the expression of fenofibrate down regulation of VEGFC. Conclusion 1.CoCl2 induced hypoxia in RPE cells in vitro leads to the up regulation of VEGFC and VEGFR-3 expression, and fenofibrate can inhibit VEGF under the hypoxia state of RPE cells. The expression of C and VEGFR-3 was up-regulated, and the concentration of VEGFC and VEGFR-3 in the supernatant of cell culture after 2.RPE hypoxia increased, and the supernatant had the role of promoting proliferation, migration and capillary formation of HUVEC cells, suggesting that RPE cells played an important role in the formation of CNV caused by hypoxia, and fenofibrate could inhibit the hypoxia of RPE cells. After the up regulation of VEGFC and VEGFR-3, it is possible to indirectly influence the activity of RPE to indirectly affect the activity of HUVEC cells to inhibit the formation of the neovascularization, which is mainly expressed in the retinal ganglion cell layer above the laser injured region of the BN rat, and VEGFR-3 is mainly expressed in the choroidal damage area at the laser spot; 4. Fenofibrate can downregulate the expression of VEGFC in the choroid and retina, and the down-regulation of VEGFR-3 in choroid may be indirectly mediated by VEGFC.

【學位授予單位】：昆明醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R-332;R773.4

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