多重酪氨酸激酶受体抑制剂decorin对高糖低氧条件下血-视网膜内屏障的保护作用及机制研究
发布时间：2018-07-25 10:59
【摘要】：目的研究核心蛋白多糖(decorin)对高糖低氧条件下血-视网膜内屏障的保护作用及机制。方法培养人脐静脉内皮细胞,采用细胞计数试剂盒检测不同浓度decorin对人脐静脉内皮细胞存活率的影响;高糖低氧条件下(25 mmol·L~(-1)右旋葡萄糖+100μmol·L~(-1)CoCl_2)孵育人脐静脉内皮细胞,通过ELISA检测不同浓度decorin处理后,不同时间点人脐静脉内皮细胞分泌血管内皮生长因子水平。将细胞分为正常对照组(5.5 mmol·L~(-1)右旋葡萄糖)、高糖低氧组(25 mmol·L~(-1)右旋葡萄糖+100μmol·L~(-1)CoCl_2)、甘露醇对照组(5.5 mmol·L~(-1)右旋葡萄糖+19.5 mmol·L~(-1)甘露醇)及decorin处理组(25 mmol·L~(-1)右旋葡萄糖+100μmol·L~(-1)CoCl_2+100 nmol·L~(-1)decorin),通过检测跨内皮细胞电阻(transendothelial electrical resistance,TER)、异硫氰酸荧光素标记的右旋糖酐(fluorescein isothiocyanate,FITC-dextran)的渗漏率来评估单层人脐静脉内皮细胞的屏障功能;Western blotting检测内皮细胞间紧密连接蛋白(claudin-5、occludin、ZO~(-1))及p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)的磷酸化水平。结果细胞计数试剂盒检测发现,10 nmol·L~(-1)、50 nmol·L~(-1)、100 nmol·L~(-1)、200 nmol·L~(-1)decorin处理人脐静脉内皮细胞24 h后,各组人脐静脉内皮细胞的存活率均大于90%,差异无统计学意义(均为P0.05)。在接种后14 d,单层人脐静脉内皮细胞的TER达到最大值且趋于稳定为(170.67±9.07)Ω。与正常对照组比较,高糖低氧处理48 h后,高糖低氧组的TER显著降低至(97.33±6.11)Ω,而decorin组的TER能够维持在(157.67±11.72)Ω,与高糖低氧组相比,差异有统计学意义(P0.05)。高糖低氧处理48 h后,高糖低氧组FITC-dextran的渗透性明显增加,488 nm处的吸光度值为正常对照组的(2.12±0.07)倍(P0.05)。加入100 nmol·L~(-1)的decorin处理后,FITC-dextran的渗透性明显减少,吸光度值是正常对照组的(1.16±0.03)倍,与高糖低氧组的差异有统计学意义(P0.05)。高糖低氧处理48 h后,高糖低氧组紧密连接蛋白claudin-5表达量为0.38±0.05、occludin为0.43±0.02及ZO~(-1)为0.25±0.02与正常对照组表达量0.72±0.05、0.90±0.01和0.75±0.02相比,差异均有统计学意义(均为P0.05)。decorin组claudin-5、occludin及ZO~(-1)表达量分别为0.65±0.08、0.87±0.03和0.60±0.01,与高糖低氧组相比,差异均有统计学意义(均为P0.05)。高糖低氧组p-p38 MAPK/p38 MAPK的比值增加,为0.88±0.02,而decorin组p-p38 MAPK/p38 MAPK的比值(0.58±0.04)接近正常对照组水平(0.56±0.02),与高糖低氧组比较,差异有统计学意义(P0.05)。结论 decorin能够保护高糖低氧条件下单层人脐静脉内皮细胞的屏障功能并下调p38 MAPK的表达水平,是治疗糖尿病视网膜病变的可能药物。
[Abstract]:Objective to study the protective effect and mechanism of core proteoglycan (decorin) on blood-retinal barrier under high glucose and hypoxia conditions. Methods cultured human umbilical vein endothelial cells (HUVECs), the effects of different concentrations of decorin on the survival rate of human umbilical vein endothelial cells (HUVECs) were detected by cell count kit, and human umbilical vein endothelial cells (HUVEC) were incubated with 25 mmol L-1 dextrose (100 渭 mol L ~ (-1) CoCl_2) under high glucose and hypoxia conditions. The levels of vascular endothelial growth factor (VEGF) secreted by human umbilical vein endothelial cells (HUVECs) at different time points after different concentrations of decorin were detected by ELISA. The cells were divided into normal control group (5.5 mmol L ~ (-1) dextrose), high glucose hypoxia group (25 mmol L ~ (-1) dextrose 100 渭 mol L ~ (-1) CoCl_2), mannitol control group (5.5 mmol L ~ (-1) dextrose 19.5 mmol L ~ (-1) mannitol) and decorin treated group (25 mmol L ~ (-1) dextrose 100 渭 mol L ~ (-1) CoCl_2 100 nmol L ~ (-1) decorin),). The leakage rate of fluorescein isothiocyanate FITC-dextran (fluorescein isothiocyanate FITC-dextran) was measured to evaluate the barrier function of monolayer human umbilical vein endothelial cells. Western blotting was used to detect claudin-5occludinZO-1 and p38 mitogen. Phosphorylation level of p38 mitogen-activated protein kinase p38 MAPK. Results the cell count kit showed that the survival rate of human umbilical vein endothelial cells (HUVECs) in 10 nmol L-1 50 nmol L-1 100 nmol L-1 decorin for 24 h was higher than that in 90 cells (P0.05). On the 14th day after inoculation, the TER of monolayer human umbilical vein endothelial cells reached the maximum value and tended to be stable to (170.67 卤9.07) 惟. Compared with the normal control group, the TER of the high glucose hypoxia group decreased significantly to (97.33 卤6.11) 惟 after 48 h treatment, while the TER of the decorin group was maintained at (157.67 卤11.72) 惟. The difference was statistically significant compared with the high glucose hypoxia group (P0.05). After treatment with high glucose and hypoxia for 48 h, the permeability of FITC-dextran in high glucose hypoxia group was significantly increased by (2.12 卤0.07) times than that in control group at 488nm (P0.05). The osmotic property of FITC-dextran was significantly decreased after the addition of 100 nmol L ~ (-1) decorin, and the absorbance was 1.16 卤0.03 times of that of the normal control group, which was significantly different from that of the high glucose hypoxia group (P0.05). After treatment with high glucose and hypoxia for 48 h, the expression levels of claudin-5 and ZO-1 in high glucose hypoxia group were 0.38 卤0.05Ocludin 0.43 卤0.02 and 0.25 卤0.02, respectively, compared with 0.72 卤0.05U 0.90 卤0.01and 0.75 卤0.02 in control group. The expression levels of claudin-5occludin and ZO-1 in decorin group were 0.65 卤0.080.87 卤0.03 and 0.60 卤0.01, respectively, which were significantly higher than those in high glucose and hypoxia group (P0.05). The ratio of p-p38 MAPK/p38 MAPK in high glucose hypoxia group was 0.88 卤0.02, while that in decorin group was (0.58 卤0.04) close to that in normal control group (0.56 卤0.02), which was significantly higher than that in high glucose hypoxia group (P0.05). Conclusion decorin can protect the barrier function and down-regulate the expression of p38 MAPK in monolayer human umbilical vein endothelial cells under high glucose and hypoxia conditions. It is a possible drug for the treatment of diabetic retinopathy.
【作者单位】： 上海交通大学附属第六人民医院眼科;郑州大学第一附属医院眼科;
【基金】：国家自然科学基金资助(编号:81400414、81271031、81600736)~~
【分类号】：R774.1
[Abstract]:Objective to study the protective effect and mechanism of core proteoglycan (decorin) on blood-retinal barrier under high glucose and hypoxia conditions. Methods cultured human umbilical vein endothelial cells (HUVECs), the effects of different concentrations of decorin on the survival rate of human umbilical vein endothelial cells (HUVECs) were detected by cell count kit, and human umbilical vein endothelial cells (HUVEC) were incubated with 25 mmol L-1 dextrose (100 渭 mol L ~ (-1) CoCl_2) under high glucose and hypoxia conditions. The levels of vascular endothelial growth factor (VEGF) secreted by human umbilical vein endothelial cells (HUVECs) at different time points after different concentrations of decorin were detected by ELISA. The cells were divided into normal control group (5.5 mmol L ~ (-1) dextrose), high glucose hypoxia group (25 mmol L ~ (-1) dextrose 100 渭 mol L ~ (-1) CoCl_2), mannitol control group (5.5 mmol L ~ (-1) dextrose 19.5 mmol L ~ (-1) mannitol) and decorin treated group (25 mmol L ~ (-1) dextrose 100 渭 mol L ~ (-1) CoCl_2 100 nmol L ~ (-1) decorin),). The leakage rate of fluorescein isothiocyanate FITC-dextran (fluorescein isothiocyanate FITC-dextran) was measured to evaluate the barrier function of monolayer human umbilical vein endothelial cells. Western blotting was used to detect claudin-5occludinZO-1 and p38 mitogen. Phosphorylation level of p38 mitogen-activated protein kinase p38 MAPK. Results the cell count kit showed that the survival rate of human umbilical vein endothelial cells (HUVECs) in 10 nmol L-1 50 nmol L-1 100 nmol L-1 decorin for 24 h was higher than that in 90 cells (P0.05). On the 14th day after inoculation, the TER of monolayer human umbilical vein endothelial cells reached the maximum value and tended to be stable to (170.67 卤9.07) 惟. Compared with the normal control group, the TER of the high glucose hypoxia group decreased significantly to (97.33 卤6.11) 惟 after 48 h treatment, while the TER of the decorin group was maintained at (157.67 卤11.72) 惟. The difference was statistically significant compared with the high glucose hypoxia group (P0.05). After treatment with high glucose and hypoxia for 48 h, the permeability of FITC-dextran in high glucose hypoxia group was significantly increased by (2.12 卤0.07) times than that in control group at 488nm (P0.05). The osmotic property of FITC-dextran was significantly decreased after the addition of 100 nmol L ~ (-1) decorin, and the absorbance was 1.16 卤0.03 times of that of the normal control group, which was significantly different from that of the high glucose hypoxia group (P0.05). After treatment with high glucose and hypoxia for 48 h, the expression levels of claudin-5 and ZO-1 in high glucose hypoxia group were 0.38 卤0.05Ocludin 0.43 卤0.02 and 0.25 卤0.02, respectively, compared with 0.72 卤0.05U 0.90 卤0.01and 0.75 卤0.02 in control group. The expression levels of claudin-5occludin and ZO-1 in decorin group were 0.65 卤0.080.87 卤0.03 and 0.60 卤0.01, respectively, which were significantly higher than those in high glucose and hypoxia group (P0.05). The ratio of p-p38 MAPK/p38 MAPK in high glucose hypoxia group was 0.88 卤0.02, while that in decorin group was (0.58 卤0.04) close to that in normal control group (0.56 卤0.02), which was significantly higher than that in high glucose hypoxia group (P0.05). Conclusion decorin can protect the barrier function and down-regulate the expression of p38 MAPK in monolayer human umbilical vein endothelial cells under high glucose and hypoxia conditions. It is a possible drug for the treatment of diabetic retinopathy.
【作者单位】： 上海交通大学附属第六人民医院眼科;郑州大学第一附属医院眼科;
【基金】：国家自然科学基金资助(编号:81400414、81271031、81600736)~~
【分类号】：R774.1
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