中华眼底病杂志

中华眼底病杂志

高糖缺氧环境下转甲状腺素蛋白对人视网膜血管内皮细胞生长抑制作用机制

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目的 探讨高糖缺氧环境下转甲状腺素蛋白(TTR)对人视网膜血管内皮细胞(hREC)生长抑制作用的机制。 方法 将hREC分为正常组、高糖组、正常缺氧组、高糖缺氧组、正常+TTR组、高糖+TTR组、正常缺氧+TTR组及高糖缺氧+TTR组,各组细胞均置于Dulbecco改良Eagle培养基中培养。正常组、正常缺氧组培养基中加入5.5 mmol/L葡萄糖;高糖组、高糖缺氧组培养基中加入25.0 mmol/L葡萄糖;正常缺氧组、高糖缺氧组培养基中加入200 μmol/L氯化钴;正常+TTR组、高糖+TTR组、正常缺氧+TTR组及高糖缺氧+TTR组除高糖及缺氧诱导外,于细胞贴壁后再加入4 μmol/L TTR。采用流式细胞仪检测各组细胞凋亡率;蛋白免疫印迹法(Western blot)检测各组细胞磷酸激酶B(Akt)、磷酸化Akt(p-Akt)、B细胞淋巴瘤/白血病-2(Bcl-2)、内皮型一氧化氮合成酶(eNOS)及Bcl-2相关X蛋白(Bax)的蛋白表达量。 结果 正常缺氧组较正常组(χ2=25.360)、高糖缺氧组较高糖组(χ2=17.400)、高糖缺氧+TTR组较高糖缺氧组(χ2=9.900)均表现为细胞凋亡率明显增加,差异有统计学意义(P<0.05)。正常组与高糖组(χ2=0.010)、正常组与正常+TTR组、高糖组与高糖+TTR组、正常缺氧组与正常缺氧+TTR组之间细胞凋亡率比较,差异均无统计学意义(P>0.05)。Western blot检测结果显示,各组Akt蛋白表达比较,差异无统计学意义(F=2.450,P>0.05)。正常缺氧组较正常组(t=9.406、5.306、4.819、−4.503)、高糖缺氧组较高糖组(t=8.877、7.723、6.500、−14.646)均表现为p-Akt、eNOS、Bcl-2蛋白表达量明显减少,Bax蛋白表达量明显增加,差异有统计学意义(P<0.05)。与正常缺氧组比较,正常缺氧+TTR组p-Akt蛋白表达量增加(t=−5.024,P<0.05),差异有统计学意义(P<0.05);eNOS、Bcl-2、Bax蛋白表达量无明显变化,差异无统计学意义(t=−2.235、−2.656、−0.272,P>0.05)。与高糖缺氧组比较,高糖缺氧+TTR组p-Akt、Bcl-2蛋白表达量明显减少(t=4.355、4.308),Bax蛋白表达量明显增加(t=−4.311),差异有统计学意义(P<0.05);eNOS蛋白表达量无明显变化,差异无统计学意义(t=−1.590,P>0.05)。高糖组与正常组、正常+TTR组与正常组、高糖+TTR组与高糖组之间p-Akt、eNOS、Bcl-2、Bax蛋白表达量比较,差异均无统计学意义(P>0.05)。 结论 高糖缺氧环境下TTR对hREC生长抑制作用的机制与Akt/Bcl-2/Bax信号通路有关,而与Akt/eNOS信号通路无关。

Objective To explore repressive effects of transthyretitin (TTR) on the growth of human retinal endothelial cells (hREC) under high glucose and hypoxia environment. Methods hRECs were divided into 8 groups, including normal glucose group (5.5 mmol/L glucose), hypoxia group, high glucose group (25.0 mmol/L glucose), high glucose and hypoxia group, normal glucose group+TTR, normal glucose and hypoxia group+TTR, high glucose group+TTR, high glucose and hypoxia group+TTR. Flow cytometry was used to analyze cellular apoptosis. The expression level of Akt, p-Akt, eNOS, Bcl-2 and Bax protein were measured by Western blot. Results Hypoxia could induce apoptosis as the apoptosis rate of normal and hypoxia group was higher than normal group (χ2=25.360, P<0.05), high glucose and hypoxia group was higher that high glucose group (χ2=17.400, P<0.05). The cell apoptosis rate of high glucose and hypoxia group+TTR were increased significantly as compared with high glucose and hypoxia group (χ2=9.900, P<0.05). There was no statistically significant difference on the cell apoptosis rate between normal group and high glucose group, normal group+TTR and normal group, high glucose group+TTR and high glucose group, normal and hypoxia group+TTR and normal and hypoxia group (P>0.05). Western blot showed that the expression of Akt did not change significantly in all eight groups(F=2.450, P>0.05). Compared to normal group, the expression of p-Akt, eNOS, Bcl-2 in normal and hypoxia group were decreased (t=9.406, 5.306, 4.819), and the expression of Bax (t=−4.503) was increased (P<0.05). Compared to high glucose group, same trend was found in high glucose and hypoxia group (t=8.877, 7.723, 6.500, −14.646; P<0.05). The expression of p-Akt in normal and hypoxia group+TTR was higher than normal and hypoxia group (t=−5.024, P<0.05) , but there was no difference on the expression of eNOS, Bcl-2, Bax between these two groups (t=−2.235, −2.656, −0.272; P>0.05). Compared to high glucose and hypoxia group, the expression of p-Akt and Bcl-2 in high glucose and hypoxia group+TTR were decreased (t=4.355, 4.308; P<0.05), the expression of Bax was increased (t=−4.311, P<0.05), and there was no difference on the expression of eNOS between these two groups (t=−1.590, P>0.05). There was no statistically significant difference in the expression of p-Akt, eNOS, Bcl-2, Bax between high glucose group and normal group (t=−3.407, −4.228, −4.302, −2.076; P>0.05), normal group+TTR and normal group (t=−4.245, −4.298, −2.816, −1.326; P>0.05), high glucose group+TTR and high glucose group (t=4.016, −0.784, 0.707, −0.328; P>0.05). Conclusion Under high glucose and hypoxia, transthyretitin suppress the growth of hREC through Akt/Bcl-2/Bax, but not Akt/eNOS signaling pathway.

关键词: 视网膜血管/细胞学; 内皮细胞/生理学; 前白蛋白/药物作用

Key words: Retinal Vessels/cytology; Endothelial cells/physiology; Prealbumin/drug effects

引用本文: 殷晓雯, 邵珺, 邹健, 殷莹, 胡亚玲, 李正, 宗达, 陈璇, 庄淼, 谭澄烨, 刘艳, 姚勇. 高糖缺氧环境下转甲状腺素蛋白对人视网膜血管内皮细胞生长抑制作用机制. 中华眼底病杂志, 2017, 33(5): 523-526. doi: 10.3760/cma.j.issn.1005-1015.2017.05.019 复制

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