论文部分内容阅读
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)100μmol/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-1αmRNA的表达水平较A组明显升高(P<0.01),B、C、D 3组间HIF-1αmRNA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。
OBJECTIVE: To investigate the effect of PEDF on the secretion of vascular endothelial growth factor (VEGF) from human bronchial epithelial cells (HBE) in hypoxia and to explore its relationship with hypoxia inducible factor 1α (HIF-1α) Sex. Methods: HBE cells cultured in vitro were used for experiments from the third to the seventh generation, and cobalt chloride (CoCl2) was used to simulate hypoxia state at 100μmol / L. The experiment was divided into four groups: the normal control group (group A), cobalt chloride intervention group (group B), cobalt chloride + PEDF 50 ng / ml intervention group (group C) and cobalt chloride + PEDF 200 ng / ml intervention Group (Group D). The expression of HIF-1α and VEGF in each group were detected by RT-PCR. The expression of VEGF protein in each group was detected by ELISA and Western blot. The expression of HIF-1α in each group was detected by immunofluorescence staining (IF) Protein expression in cytoplasm and nucleus. Results: ① The mRNA level of VEGF in group B was significantly higher than that in group A [(8.56 ± 0.67) times in group A, P <0.01]. VEGF mRNA level in group C and group D was significantly lower than that in group B (P <0.01) ; The expression level of HIF-1αmRNA in group B was significantly higher than that in group A (P <0.01), but there was no significant difference in HIF-1αmRNA in group B, C and D; ② The expression of VEGF in group B was significantly higher than that in group B (1 370.10 ± 42.98) pg / ml] was significantly higher than that of group A [(670.00 ± 23.35) pg / ml] Group [(646.47 ± 22.70) pg / ml], the difference was statistically significant (P <0.01). (3) Western blot showed that the protein expression of VEGF in group B was (3.99 ± 0.37) times of that of group A, the difference was statistically significant (P <0.01); the protein expression of VEGF in group C and group D was significantly lower than that in group B There was statistical significance (P <0.05). ④IF results showed that the expression level of HIF-1α in B group and intranuclear metastasis were significantly higher than those in A and D groups. CONCLUSION: PEDF can inhibit the expression of VEGF in HBE cells under hypoxic condition, and the inhibitory effect may be related to the regulation of HIF-1α by PEDF.