【摘要】：目的研究核心蛋白多糖(decorin)對高糖低氧條件下血-視網(wǎng)膜內(nèi)屏障的保護(hù)作用及機(jī)制。方法培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞,采用細(xì)胞計數(shù)試劑盒檢測不同濃度decorin對人臍靜脈內(nèi)皮細(xì)胞存活率的影響;高糖低氧條件下(25 mmol·L~(-1)右旋葡萄糖+100μmol·L~(-1)CoCl_2)孵育人臍靜脈內(nèi)皮細(xì)胞,通過ELISA檢測不同濃度decorin處理后,不同時間點人臍靜脈內(nèi)皮細(xì)胞分泌血管內(nèi)皮生長因子水平。將細(xì)胞分為正常對照組(5.5 mmol·L~(-1)右旋葡萄糖)、高糖低氧組(25 mmol·L~(-1)右旋葡萄糖+100μmol·L~(-1)CoCl_2)、甘露醇對照組(5.5 mmol·L~(-1)右旋葡萄糖+19.5 mmol·L~(-1)甘露醇)及decorin處理組(25 mmol·L~(-1)右旋葡萄糖+100μmol·L~(-1)CoCl_2+100 nmol·L~(-1)decorin),通過檢測跨內(nèi)皮細(xì)胞電阻(transendothelial electrical resistance,TER)、異硫氰酸熒光素標(biāo)記的右旋糖酐(fluorescein isothiocyanate,FITC-dextran)的滲漏率來評估單層人臍靜脈內(nèi)皮細(xì)胞的屏障功能;Western blotting檢測內(nèi)皮細(xì)胞間緊密連接蛋白(claudin-5、occludin、ZO~(-1))及p38絲裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)的磷酸化水平。結(jié)果細(xì)胞計數(shù)試劑盒檢測發(fā)現(xiàn),10 nmol·L~(-1)、50 nmol·L~(-1)、100 nmol·L~(-1)、200 nmol·L~(-1)decorin處理人臍靜脈內(nèi)皮細(xì)胞24 h后,各組人臍靜脈內(nèi)皮細(xì)胞的存活率均大于90%,差異無統(tǒng)計學(xué)意義(均為P0.05)。在接種后14 d,單層人臍靜脈內(nèi)皮細(xì)胞的TER達(dá)到最大值且趨于穩(wěn)定為(170.67±9.07)Ω。與正常對照組比較,高糖低氧處理48 h后,高糖低氧組的TER顯著降低至(97.33±6.11)Ω,而decorin組的TER能夠維持在(157.67±11.72)Ω,與高糖低氧組相比,差異有統(tǒng)計學(xué)意義(P0.05)。高糖低氧處理48 h后,高糖低氧組FITC-dextran的滲透性明顯增加,488 nm處的吸光度值為正常對照組的(2.12±0.07)倍(P0.05)。加入100 nmol·L~(-1)的decorin處理后,FITC-dextran的滲透性明顯減少,吸光度值是正常對照組的(1.16±0.03)倍,與高糖低氧組的差異有統(tǒng)計學(xué)意義(P0.05)。高糖低氧處理48 h后,高糖低氧組緊密連接蛋白claudin-5表達(dá)量為0.38±0.05、occludin為0.43±0.02及ZO~(-1)為0.25±0.02與正常對照組表達(dá)量0.72±0.05、0.90±0.01和0.75±0.02相比,差異均有統(tǒng)計學(xué)意義(均為P0.05)。decorin組claudin-5、occludin及ZO~(-1)表達(dá)量分別為0.65±0.08、0.87±0.03和0.60±0.01,與高糖低氧組相比,差異均有統(tǒng)計學(xué)意義(均為P0.05)。高糖低氧組p-p38 MAPK/p38 MAPK的比值增加,為0.88±0.02,而decorin組p-p38 MAPK/p38 MAPK的比值(0.58±0.04)接近正常對照組水平(0.56±0.02),與高糖低氧組比較,差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論 decorin能夠保護(hù)高糖低氧條件下單層人臍靜脈內(nèi)皮細(xì)胞的屏障功能并下調(diào)p38 MAPK的表達(dá)水平,是治療糖尿病視網(wǎng)膜病變的可能藥物。
[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 鹵9.07) 惟. Compared with the normal control group, the TER of the high glucose hypoxia group decreased significantly to (97.33 鹵6.11) 惟 after 48 h treatment, while the TER of the decorin group was maintained at (157.67 鹵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 鹵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 鹵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 鹵0.05Ocludin 0.43 鹵0.02 and 0.25 鹵0.02, respectively, compared with 0.72 鹵0.05U 0.90 鹵0.01and 0.75 鹵0.02 in control group. The expression levels of claudin-5occludin and ZO-1 in decorin group were 0.65 鹵0.080.87 鹵0.03 and 0.60 鹵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 鹵0.02, while that in decorin group was (0.58 鹵0.04) close to that in normal control group (0.56 鹵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.
【作者單位】： 上海交通大學(xué)附屬第六人民醫(yī)院眼科;鄭州大學(xué)第一附屬醫(yī)院眼科;
【基金】：國家自然科學(xué)基金資助(編號:81400414、81271031、81600736)~~
【分類號】：R774.1

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