二甲双胍联合康柏西普对VEGF诱导的内皮细胞改变的协同作用

发布时间：2018-02-28 23:04

本文关键词： 糖尿病黄斑水肿康柏西普二甲双胍血-视网膜屏障 VEGF　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的糖尿病黄斑水肿是成人视力受损的重要原因之一,其发病机制主要与血-视网膜屏障受损有关。抗VEGF药物玻璃体腔注射已成为治疗该病的重要手段之一,但并非所有患者对该药物反映良好。二甲双胍作为一种传统降糖药,近年来在抗肿瘤、抗衰老等领域研究颇多。本研究旨在探究康柏西普与二甲双胍的联合应用对视网膜血管内皮细胞的协同作用及可能机制,为临床治疗提供理论基础。方法回顾性分析2016年1月~2016年12月于天津医科大学眼科医院因糖尿病黄斑水肿行玻璃体腔注射康柏西普的患者,对比服用二甲双胍组与未服用二甲双胍组视网膜中心凹厚度(CRT)改变情况。体外培养RF/6A,将细胞分为五个组:对照组、VEGF刺激组、VEGF+康柏西普刺激组、VEGF+二甲双胍刺激组、VEGF+康柏西普+二甲双胍刺激组。设置VEGF、康柏西普及二甲双胍浓度梯度,用1640完全培养基将其稀释,利用MTT比色法选取药物最佳浓度及实验观测时点;用MTT细胞增殖实验检测两种药物联合应用对VEGF诱导的RF/6A细胞增殖的影响;通过细胞划痕实验观察两种药物联合应用对VEGF诱导的RF/6A细胞迁移能力的影响;通过FQRT-PCR检测药物作用下RF/6A细胞血管内皮生长因子受体2(VEGFR2)、蛋白激酶C-α(PKC-α)和蛋白激酶C-β(PKC-β)的基因表达情况。结果10例(12眼)患者纳入数据分析,对比术后3月CRT恢复率,二甲双胍组明显优于对照组,差异有统计学意义(P0.05)。50ng/ml为VEGF的最佳刺激浓度;2.5ug/ml为康柏西普最佳给药浓度;2.0mmol/L为二甲双胍最佳给药浓度;72h为最佳观测时点。MTT细胞增殖实验结果显示:与对照组(0.5136±0.0311)及VEGF刺激组(0.8179±0.0288)相比,干预组细胞增殖明显受到抑制(P0.001);联合用药组(0.2991±0.0323)与单独应用康柏西普组(0.3408±0.0274)相比,OD值显著降低(P0.05)。细胞划痕实验结果显示:与对照组相比,VEGF组细胞迁移率显著升高(P0.001);与VEGF组相比,干预组细胞迁移率显著降低(P0.05);12h时,联合用药组(0.1085±0.0141)与康柏西普组(0.1324±0.0379)相比,细胞迁移率显著降低(P0.05)。FQRT-PCR实验结果显示:与对照组相比,VEGF组VEGFR2、PKC-α和PKC-β的基因表达量均显著升高(P0.01);联合用药组与VEGF组相比,VEGFR2、PKC-α和PKC-β的基因表达量均显著降低(P0.05);联合用药组与康柏西普组相比,PKC-α的基因表达量显著降低(P0.05)。结论因糖尿病黄斑水肿行玻璃体腔注射康柏西普的患者,二甲双胍对药效时间有一定积极作用。联合应用康柏西普及二甲双胍,可以抑制VEGF所引起的血管内皮细胞生物学改变及相关基因的表达,且作用明显优于单独应用康柏西普。
[Abstract]:Objective Diabetic macular edema is one of the most important causes of visual impairment in adults, and its pathogenesis is mainly related to the damage of blood-retina barrier. Antidiabetic intravitreal injection of VEGF has become one of the important methods for the treatment of this disease. But not all patients reacted well to the drug. Metformin, as a traditional hypoglycemic drug, has been working on antitumor drugs in recent years. The aim of this study was to explore the synergistic effect and possible mechanism of Compactopril and metformin on retinal vascular endothelial cells (RVEC). Methods from January 2016 to December 2016, the patients with macular edema in Tianjin Medical University were treated with intravitreal injection of Combortopril in the ophthalmology hospital of Tianjin Medical University. The changes of retinal foveal thickness (CRT) were compared between metformin group and non-metformin group. RFR / 6A was cultured in vitro. Compacipe metformin stimulation group. Set up VEGF, Compaq universal metformin concentration gradient, The best concentration and time point were selected by MTT colorimetry, and the effects of two drugs on the proliferation of RF/6A cells induced by VEGF were detected by MTT cell proliferation assay. The effects of combined use of two drugs on the migration of RF/6A cells induced by VEGF were observed by cell scratch assay. FQRT-PCR was used to detect the gene expression of vascular endothelial growth factor receptor (VEGFR2A), protein kinase C- 伪 (PKC- 伪) and protein kinase C- 尾 (PKC- 尾) in RF/6A cells. Results 10 patients (12 eyes) were enrolled in the data analysis, and the recovery rate of CRT was compared on March. Metformin group was superior to control group. The difference was statistically significant (P < 0.05) .50ng / ml was the best stimulating concentration of VEGF. The best concentration of VEGF was 2.5ugr / ml, the best dose of Compactopril was 2.0 mmol / L, the best concentration of metformin was 72 hours. The results of cell proliferation test showed that compared with the control group (0.5136 卤0.0311) and the VEGF group (0.8179 卤0.0288), the difference was significant. The cell proliferation was significantly inhibited in the intervention group (P 0.001) and the OD value in the combined treatment group (0.2991 卤0.0323) was significantly lower than that in the control group (0.3408 卤0.0274). The cell scratch test showed that the cell migration rate of the VEGF group was significantly higher than that of the control group, and that of the VEGF group was significantly higher than that of the control group. The cell migration rate in the intervention group was significantly lower than that in the control group (0.1085 卤0.0141) and that in the control group (0.1324 卤0.0379) at 12h. The results of reverse transcription polymerase chain reaction (RT-PCR) showed that the expression of VEGFR2PKC- 伪 and PKC- 尾 in VEGFR2PKC- 伪 and PKC- 尾 were significantly increased in VEGFR2PKC- 伪 and PKC- 尾, and the expression of PKC- 伪 and PKC- 尾 in VEGFR2PKC- 伪 and PKC- 尾 in combination group were significantly lower than those in VEGF group, and the expression of PKC- 伪 and PKC- 尾 in combination group was significantly lower than that in VEGF group. The expression of PKC- 伪 gene decreased significantly compared with the control group. Conclusion the patients with diabetic macular edema treated by intravitreous injection of Compactopril, and the patients with diabetic macular edema were treated with intravitreous injection of Compactopril, and the expression of PKC- 伪 decreased significantly. Metformin had a positive effect on the time of pharmacodynamics. The combination of Compactyl and metformin could inhibit the biological changes of vascular endothelial cells and the expression of related genes induced by VEGF, and the effect was obviously better than that of Compactopril alone.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R774.5

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