摘要
观察在脯氨酸羟化酶抑制剂(PHI)干预下,脯氨酸羟化酶2(PHD 2)在缺氧环境下大鼠视网膜神经胶质细胞中的表达变化,探讨其与眼内相关增生因子表达的关系。原代培养大鼠视网膜神经胶质细胞,鉴定后传代培养,并分为3组:正常对照组(control)、缺氧组(hypoxia)、干预组(intervention)。正常对照组不做特殊处理;缺氧组加入200μmol/L缺氧诱导剂氯化钴(CoCl2);干预组加入500μmol/L的脯氨酸羟化酶抑制剂。免疫荧光化学染色法观察各组PHD 2和血管内皮生长因子(VEGF)蛋白表达,RT-PCR检测PHD 2、VEGF mRNA水平,四甲基偶氮唑蓝比色法(MTT法)检测各组细胞增殖活性,Annexin V-FITC鉴定各组细胞凋亡情况。对照组视网膜神经胶质细胞中PHD 2和VEGF的表达比较弱,呈弱荧光染色;缺氧组视网膜神经胶质细胞中PHD 2及VEGF的表达明显增强,呈强红色荧光及强绿色荧光;干预组PHD 2及VEGF的表达明显低于缺氧组。RT-PCR分析结果表明:对照组视网膜神经胶质细胞中PHD 2和VEGF mRNA都呈弱表达;缺氧组视网膜神经胶质细胞中PHD 2和VEGF mRNA表达量明显增多;干预组中PHD 2、VEGF mRNA表达量较对照组轻度增多,但较缺氧组明显减少。缺氧组视网膜神经胶质细胞的增殖活性明显高于正常对照组(P<0.001);干预组视网膜神经胶质细胞的增殖活性高于正常对照组(P=0.001);但是干预组视网膜神经胶质细胞增殖活性明显低于缺氧组(P=0.001)。干预组和对照组视网膜神经胶质细胞的凋亡明显高于缺氧组(P<0.001);干预组视网膜神经胶质细胞的凋亡高于正常对照组(P<0.001)。缺氧环境可诱导视网膜神经胶质细胞中PHD 2、VEGF mRNA的表达增高,刺激神经胶质细胞的增殖,减缓细胞凋亡;脯氨酸羟化酶抑制剂可有效抑制缺氧环境下PHD 2增高引起的神经胶质细胞的增殖和VEGF mRNA的表达。
The research was designed to investigate the effect of proline hydroxylase inhibitor(PHI)on the expression of proline hydroxylase 2(PHD 2)in rat retinal glial cells under hypoxia and the relationship between intervention of PHI and growth factor expression in eyes.Primary rat retinal glial cells were identified and sub-cultured,then divided into three groups:control group(control,cells were untreated),hypoxia group(hypoxia,200μmol/L of cobalt chloride was used to induce hypoxia)and intervention group(intervention,500μmol/L of PHI was added).The immunofluorescence staining was used to assay the protein levels of PHD 2 and vascular endothelial growth factor(VEGF)and the real time RT-PCR was used to detect the mRNA levels of PHD 2 and VEGF.To measure cell proliferation and cell apoptosis,MTT assay and Annexin V-FITC kit were applied respectively.The results revealed that the PHD 2 and VEGF were weakly expressed in the control group,showing weak fluorescence staining,while they were over-expressed in the hypoxia group,showing strengthened red and green fluorescence staining,and PHI treatment significantly decreased the expression of PHD 2 and VEGF compared with the hypoxia group.The real time RT-PCR showed that the mRNAs of PHD 2 and VEGF were low level expressed in control group,while they were obviously increased under the hypoxia condition,but were significantly decreased by PHI intervention even though they were slightly higher than those in the control group.MTT assay gave out that hypoxia treatment slightly increased the proliferative activity of retinal glial cells compared with the control(P<0.001),but it was significantly decreased after intervention with PHI(P=0.001),even though it was still higher than that in the control group(P=0.001).The determination of Annexin V-FITC kit found that both of the intervention group and the control group showed a significantly higher rate of cell apoptosis than the hypoxia group(P<0.001),while the apoptosis rate of the cells in the intervention group was higher than that in the control group(P<0.001).All the result demonstrated that low oxygen induced the expression of PHD 2 and VEGF mRNA in rat retinal glial cells,stimulated cell proliferation and alleviated cell apoptosis;PHI effectively inhibited both cells proliferation and VEGF mRNA expression mediated by hypoxia-induced PHD 2 over-expression.
作者
李臻
畅叶叶
路强
LI Zhen;CHANG Ye-ye;LU Qiang(Inner Mongolia People's Hospital,Hohhot 010017,China)
出处
《内蒙古师范大学学报（自然科学汉文版）》
CAS
2020年第2期148-155,共8页
Journal of Inner Mongolia Normal University(Natural Science Edition)
基金
国家自然科学基金资助项目(81260152)
内蒙古自然科学基金资助项目(2014MS0865)
内蒙古自治区人民医院院内基金项目(2016043)。
