糖尿病视网膜病变患者纤维血管膜中SIRT1的表达及其相关机制初步研究

发布时间：2018-08-23 20:20

【摘要】：[目的]糖尿病视网膜病变(Diabetic retinopathy, DR)是常见的糖尿病微血管并发症,为发达国家工作年龄人群中致盲率最高的疾病。SIRT1属于第Ⅲ组蛋白去乙酰化酶,与DR发病相关。本实验的目的为初步探讨SIRT1在DR纤维血管膜形成中的作用,研究SIRT1激动剂白藜芦醇是否对高糖培养的RPE细胞具有保护效应,及其相关机制。同时初步研究与白藜芦醇结构相近的二苯乙烯苷是否具有类似作用。 [方法]采集DR伴眼底纤维血管膜患者、非糖尿病伴特发性黄斑前膜患者眼部标本,石蜡包埋切片,行免疫组化检测两组患者SIRT1表达的差异。体外培养人视网膜色素上皮细胞,加入5.5mmol/L葡萄糖,5.5mmol/L葡萄糖加上甘露醇,25mmol/L葡萄糖,25mmol/L葡萄糖及不同浓度白藜芦醇、二苯乙烯苷,分别检测以下指标的变化情况：(1)CCK-8法检测细胞活力；(2)RT-PCR检测SIRT1、PGC-1α基因表达水平；(3)Western blot检测乙酰化NF-κB p65含量及AMPK活性；(4)ELISA法检测VEGF分泌量。 [结果]共收集DR患者纤维血管膜12例,非糖尿病患者黄斑前膜7例,纤维血管膜中SIRT1阳性率高于黄斑前膜组织(100%vs28.6%),去掉年龄因素影响后,差异具有统计学意义(p=0.021)。25mmol/L葡萄糖能降低人视网膜色素上皮细胞活力、抑制SIRT1及PGC-1α转录,激活NF-κB、抑制AMPK活性,促进VEGF分泌。加入一定浓度的白藜芦醇或二苯乙烯苷,可抑制高糖引起的细胞活力下降,减少SIRT1转录下降的水平,减少NF-κB的激活,VEGF分泌量也相应降低。同时,一定浓度的白藜芦醇及二苯乙烯苷可以减少高糖对PGC-1α转录的抑制作用,其幅度与对应的SIRT1、NF-κB改变水平并不完全一致,但与AMPK的活性变化量基本一致。 [结论]DR患者纤维血管膜中SIRT1表达升高,提示SIRT1与DR新生血管有一定关系。但高糖可抑制体外培养的人视网膜色素上皮细胞中SIRT1的表达,上述实验结果提示体外实验可能不能完全反映视网膜的生理情况；或者DR病情进展过程中,SIRT1的含量在不断变化。本实验数据提示二苯乙烯苷可能也具有SIRT1激动剂的作用,且白藜芦醇及二苯乙烯苷可能是通过SIRT1—NF-κB通路降低VEGF分泌。同时PGC-1α mRNA水平可能与AMPK活性相关。
[Abstract]:[objective] Diabetic retinopathy (Diabetic retinopathy, DR) is a common diabetic microvascular complication. SIRT1 belongs to the third histone deacetylase and is associated with the pathogenesis of DR. The purpose of this study was to investigate the role of SIRT1 in the formation of vascular membranes of DR fibers, and to investigate whether resveratrol, a SIRT1 agonist, has protective effect on RPE cells cultured with high glucose and its related mechanisms. At the same time, we also studied whether stilbene glycoside, which is similar to resveratrol, has similar effect. [methods] the ocular specimens of DR patients with fundus fibrovascular membrane and non-diabetic patients with idiopathic macular membrane were collected and embedded in paraffin sections. The difference of SIRT1 expression between the two groups was detected by immunohistochemistry. In vitro cultured human retinal pigment epithelial cells (RPE) were cultured with 5.5mmol/L glucose 5.5 mmol / L glucose + mannitol 25 mmol / L glucose and resveratrol, stilbene glucoside at different concentrations. The following indexes were detected: (1) CCK-8 method was used to detect cell viability; (2) SIRT1,PGC-1 伪 gene expression was detected by RT-PCR, (3) acetylated NF- 魏 B p65 and AMPK activity were detected by) Western blot, and (4) VEGF secretion was detected by ELISA assay. [results] A total of 12 patients with DR and 7 patients with non-diabetic macular membrane were collected. The positive rate of SIRT1 in fibrovascular membrane was higher than that in macular membrane tissue (100% vs 28.6%). The difference was statistically significant (p0. 021). 25 mmol / L glucose could decrease the activity of human retinal pigment epithelial cells, inhibit the transcription of SIRT1 and PGC-1 伪, activate NF- 魏 B, inhibit the activity of AMPK and promote the secretion of VEGF. A certain concentration of resveratrol or stilbene could inhibit the decrease of cell viability, decrease the level of SIRT1 transcription, and decrease the secretion of NF- 魏 B. At the same time, resveratrol and stilbene at a certain concentration could reduce the inhibitory effect of high glucose on PGC-1 伪 transcription, the amplitude of which was not completely consistent with the change level of SIRT1,NF- 魏 B, but it was basically consistent with the activity change of AMPK. [conclusion] the increased expression of SIRT1 in fibrovascular membrane of DR patients suggests that SIRT1 is related to DR neovascularization. However, high glucose could inhibit the expression of SIRT1 in cultured human retinal pigment epithelial cells. These results suggest that the in vitro experiments may not completely reflect the physiological conditions of the retina, or the content of SIRT1 is constantly changing during the progression of DR. These results suggest that stilbene glucoside may also have the effect of SIRT1 agonist, and resveratrol and stilbene glycoside may decrease VEGF secretion through SIRT1-NF- 魏 B pathway. At the same time, the level of PGC-1 伪 mRNA may be related to the activity of AMPK.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R587.2;R774.1

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