文章摘要
吴桢珍,查王健,陈菲菲,杨光宇,黄茂.PEDF对低氧状态下人支气管上皮细胞表达VEGF的影响[J].南京医科大学学报,2014,(4):422~426
PEDF对低氧状态下人支气管上皮细胞表达VEGF的影响
Effects of PEDF on the expression of VEGF in hypoxic human bronchial epithelial cells
投稿时间:2013-12-16  
DOI:10.7655/NYDXBNS20140404
中文关键词: 哮喘  色素上皮衍生因子  缺氧诱导因子1α  血管内皮生长因子
英文关键词: asthma  pigment epithelium-derived factor  hypoxia inducible factor-1α  vascular endothelial growth factor
基金项目:江苏高校优势学科建设工程项目
作者单位
吴桢珍 南京医科大学附属第一医院呼吸内科,江苏南京210029 
查王健 南京医科大学附属第一医院呼吸内科,江苏南京210029 
陈菲菲 南京医科大学附属第一医院呼吸内科,江苏南京210029 
杨光宇 南京医科大学附属第一医院呼吸内科,江苏南京210029 
黄茂 南京医科大学附属第一医院呼吸内科,江苏南京210029 
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中文摘要:
      目的:研究色素上皮衍生因子(PEDF)对低氧状态下人支气管上皮细胞(HBE)分泌血管内皮生长因子(VEGF)的影响,并探讨该作用与缺氧诱导因子1α(HIF-1α)的相关性?方法:体外培养的HBE细胞取第3~7代用于实验,氯化钴(CoCl2)100 ?滋mol/L模拟缺氧状态?实验分为4组:正常对照组(A组)?氯化钴干预组(B组)?氯化钴 + PEDF 50 ng/ml干预组(C组)及氯化钴 + PEDF 200 ng/ml干预组(D组)?RT-PCR法检测各组细胞HIF-1α及VEGF 在mRNA水平上的表达差异;ELISA及Western blot法检测各组细胞VEGF蛋白表达水平;细胞免疫荧光染色法(IF)检测各组细胞HIF-1α蛋白在细胞质和细胞核中的表达情况?结果:①B组VEGF的mRNA水平较A组显著升高[为A组的(8.56 ± 0.67)倍,P < 0.01],C组与D组的VEGF mRNA水平较B组明显下降(P < 0.01);B组 HIF-1α mRNA的表达水平较A组明显升高(P < 0.01),B?C?D 3组间HIF-1α mRNA无统计学差异;②细胞培养基上清中B组VEGF表达量[(1 370.10 ± 42.98)pg/ml]较A组[(670.00 ± 23.35) pg/ml]明显升高(P < 0.01),且B组与C组[(816.19 ± 37.05) pg/ml]及D组[(646.47 ± 22.70)pg/ml]比较差异有统计学意义(P < 0.01)?③Western blot结果显示B组VEGF的蛋白表达量为A组的(3.99 ± 0.37)倍,差异有统计学意义(P < 0.01);C组与D组VEGF的蛋白表达水平较B组明显下降,差异有统计学意义(P < 0.05)?④IF结果显示,B组HIF-1α的表达水平及核内转移较A组和D组明显升高?结论:PEDF对低氧状态下HBE细胞高表达的VEGF具有一定抑制作用,且该抑制作用可能与PEDF调控HIF-1α有关?
英文摘要:
      Objective:To study the effects of pigment epithelium derived factor (PEDF) on the expression of vascular endothelial growth factor(VEGF) in hypoxic human bronchial epithelial(HBE) cells,and to determine whether the expression of VEGF is associated with the expression of hypoxia-inducible factor (HIF)-1α in cultured cells. Methods:The cultured HBE cells between 3rd to 7th generations were used in the study,and 100 ?滋mol/L cobalt (II) chloride hexahydrate (CoCl2) were used to simulate anaerobic condition. There were four groups in the study:control (group A),CoCl2 100 ?滋mol/L(group B),CoCl2 100 ?滋mol/L +PEDF 50 ng/ml (group C) and CoCl2 100 ?滋mol/L +PEDF 200 ng/ml (group D). The expression of HIF-1α and VEGF mRNA were detected by real-time polymerase chain reaction (RT-PCR). The expression of VEGF in the different groups were examined by enzyme-linked immunosorbent assay(ELISA) and Western blot. Immunofluorescence (IF) was performed to test the expression of HIF-1α in cytoplasm and cytoblast. Results:①The level of VEGF mRNA was significantly elevated(8.56 ± 0.67)-fold in group B compared with that in group A (P < 0.01),which was decreased in group C and group D (P < 0.01) compared with that in group B. Compared with group A,the level of HIF-1α mRNA was elevated significantly in group B (P < 0.01). However,there were no significant differences of HIF-1α mRNA level among group B,C and D. ②The concentration of VEGF protein in cell cultural supernatant was higher in group B [(1 370.10 + 42.98)pg/ml,P < 0.01] than that in group A [(670.00 + 23.35)pg/ml]. The levels of VEGF in group C [(816.19 + 37.05)pg/ml] and group D [(646.47 + 22.70)pg/ml] were decreased compared with group B (P < 0.01). ③The level of VEGF protein detected by Western blot in group B was (3.99 ± 0.37)fold of that in group A (P < 0.01),and there were significant differences between group B and the other two groups (group C and D,P < 0.05). ④ IF showed that the level of HIF-1αprotein and the nuclear translocation of HIF-1α in group B were significantly increased compared with those in group A and D. Conclusion:PEDF could inhibit the overexpressed VEGF expression in hypoxic HBE cells,and this effect may be related to PEDF-regulated HIF-1α.
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