非诺贝特对人RPE细胞及BN大鼠CNV模型VEGF-C和VEGFR-3表达变化影响的体内、体外实验研究

发布时间：2018-05-06 11:40

本文选题：人视网膜色素上皮细胞 + 人脐静脉内皮细胞　；参考：《昆明医科大学》2016年博士论文

【摘要】：目的通过体外实验研究探讨非诺贝特对人视网膜色素上皮细胞(RPE)和人脐静脉内皮细胞(HUVEC)活力的影响及可能的机制,通过体内实验探讨激光诱导BN大鼠脉络膜新生血管形成过程中脉络膜和视网膜内VEGFC和VEGFR-3的表达变化规律及非诺贝特对脉络膜新生血管发生发展的影响作用,为眼脉络膜新生血管性相关疾病提供新的思路。方法1.将RPE和HUVEC细胞分组：RPE正常组,RPE+非诺贝特组,RPE缺氧组,RPE缺氧+非诺贝特组；HUVEC细胞正常组,]HUVEC+缺氧上清组；将健康成年BN大鼠随机分成空白对照组和实验组,实验组分为非诺贝特干预组(用药组),非干预组(实验对照组),每组以1w、2w、3w和4w为观察点。2.用超氧化物阴离子探针检测RPE缺氧情况,MTT检测细胞活力,ELISA检测RPE细胞培养液上清中VEGFC和VEGFR-3的表达；细胞划痕实验检测细胞迁移能力,细胞管腔形成实验检测]HUVEC细胞的血管形成能力,qRT-PCR和Western-blot检测RPE细胞VEGFC和VEGFR-3的mRNA和蛋白质的表达水平；用FFA检测非诺贝特干预组和非干预组激光斑处不同时间点脉络膜新生血管的渗漏面积和渗漏强度,用凝聚素B4染脉络膜铺片观察激光斑处不同时间点新生血管内皮细胞的染色程度,行BN大鼠眼球冰冻切片,用免疫荧光化学检测激光斑处脉络膜和视网膜VEGFC和VEGFR-3的分布情况,用qRT-PCR技术定量检测不同时间点各组脉络膜和视网膜内VEGFC和VEGFR-3的mRNA表达变化,用Western Blot技术,检测不同时间点各组脉络膜和视网膜内VEGFC和VEGFR-3的蛋白质表达变化。结果1.非诺贝特能抑制因缺氧引起的RPE细胞内和培养液上清中VEGFC和VEGFR-3表达上调；2.缺氧培养RPE细胞上清液可促进HUVEC细胞活力增加,细胞迁移数增多,管腔形成数增多,而非诺贝特能抑制这一过程；3.激光诱导BN大鼠脉络膜新生血管形成后,脉络膜和视网膜内均可以发现有VEGFC和VEGFR-3的表达,其中VEGFC主要表达于激光斑上方视网膜神经节细胞层内,VEGFR-3主要表达与脉络膜激光斑处的新生血管区域,正常的脉络膜和视网膜未发现有上诉两种因子的表达；4.激光诱导BN大鼠脉络膜新生血管形成后,脉络膜和视网膜表达的VEGFC和VEGFR-3呈现一定的规律性,在脉络膜和视网膜中VEGFC表达随时间的推移呈现逐渐下调的趋势,在脉络膜中VEGFR-3的表达呈现逐渐升高并持续维持于高表达水平,在视网膜中VEGFR-3基本不表达或仅有少量表达；5.非诺贝特能够明显下调激光诱导BN大鼠脉络膜新生血管形成和发展过程,其能够明显下调视网膜和脉络膜内VEGFC的表达量,脉络膜内的VEGFR-3的表达下调与非诺贝特下调VEGFC的表达量有关。结论1.CoCl2诱导体外培养的RPE细胞缺氧导致VEGFC和VEGFR-3表达上调,非诺贝特能够抑制RPE细胞缺氧状态下VEGFC和VEGFR-3的表达上调；2.RPE缺氧后的细胞培养上清液中的VEGFC和VEGFR-3浓度上升,上清液对HUVEC细胞具有促增殖、迁移和毛细血管成管活性的作用,提示RPE细胞在缺氧导致的CNV形成过程中起到了重要的作用,非诺贝特能够抑制RPE细胞缺氧后的VEGFC和VEGFR-3的上调,从而有可能通过调节RPE的功能活性来间接影响HUVEC细胞的活性从而达到抑制新生血管的形成。3.VEGFC主要表达于BN大鼠激光损伤区域上方的视网膜神经节细胞层内,VEGFR-3主要表达于激光斑处的脉络膜损伤区；4.非诺贝特能够下调脉络膜和视网膜内VEGFC的表达,对脉络膜内VEGFR-3的下调作用可能是通过VEGFC的间接影响。
[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.

【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R-332;R773.4

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