LPS和TNF-α诱导小鼠腹腔巨噬细胞HIF-1α表达的信号通路研究

发布时间：2018-04-29 19:41

本文选题：脂多糖 + 肿瘤坏死因子-α　；参考：《苏州大学》2009年硕士论文

【摘要】： 目的:研究脂多糖(LPS)和肿瘤坏死因子-α(TNF-α)对小鼠腹腔巨噬细胞缺氧诱导因子-1α(HIF-1α)表达的相关信号通路。方法:自BALB/c小鼠腹腔分离获得巨噬细胞进行体外培养,观察NF-κB抑制剂柳氮磺胺吡啶(SSZ)或一氧化氮合酶(NOS)抑制剂L-单甲基精氨酸(L-NMMA)或在两者联合作用对LPS或TNF-α诱导细胞部分信号分子表达的影响,以逆转录聚合酶链反应(RT-PCR)法检测培养细胞HIF-1αmRNA水平的变化,用免疫细胞化学染色法检测培养细胞中HIF-1α和VEGF蛋白表达的变化,用Griess反应检测培养上清中亚硝酸盐浓度的变化。结果:常氧条件下,LPS或TNF-α刺激后,巨噬细胞的HIF-1αmRNA水平无明显变化(P0.05)。巨噬细胞经LPS或TNF-α刺激10h后,HIF-1α和VEGF蛋白表达水平明显增加(P0.05);经SSZ预处理或L-NMMA作用后,巨噬细胞HIF-1α和VEGF蛋白的表达水平较单纯LPS或TNF-α刺激时明显减少(P0.05);在SSZ和L-NMMA联合作用下, HIF-1α和VEGF蛋白的表达水平明显低于LPS或TNF-α单独刺激组者(P0.05),但与SSZ或L-NMMA单独使用时的抑制效应相比,HIF-1α蛋白水平未见明显差异(P0.05),VEGF蛋白水平明显下降(P0.05)。Griess反应显示,正常对照组培养上清中亚硝酸盐的测定值较低。LPS或TNF-α刺激下,培养上清中亚硝酸盐的测定值明显升高(P0.05),L-NMMA或SSZ作用后,培养上清中亚硝酸盐的测定值较LPS或TNF-α单独刺激时者明显降低(P0.05)。常氧环境下,DETA-NO作用巨噬细胞4h后,HIF-1α和VEGF蛋白表达水平较正常对照组者明显升高(P0.05)。结论:LPS和TNF-α可通过NF-κB和NOS等转录后途径上调HIF-1α的表达, HIF-1α的表达可进一步诱导其靶基因VEGF的表达。
[Abstract]:Aim: to investigate the signal pathways associated with the expression of hypoxia-inducible factor-1 伪 (HIF-1 伪) in mouse peritoneal macrophages by lipopolysaccharide (LPS) and tumor necrosis factor- 伪 (TNF- 伪). Methods: macrophages isolated from the abdominal cavity of BALB/c mice were cultured in vitro. To observe the effects of NF- 魏 B inhibitor, sulfamyridine (SSZ) or nitric oxide synthase (NOS) inhibitor, L-NMMA-monomethyl arginine (L-NMMA) on the expression of some signal molecules induced by LPS or TNF- 伪. The level of HIF-1 伪 mRNA was detected by reverse transcriptase polymerase chain reaction (RT PCR), the expression of HIF-1 伪 and VEGF protein in cultured cells was detected by immunocytochemical staining, the nitrite concentration in culture supernatant was detected by Griess reaction. Results: the level of HIF-1 伪 mRNA in macrophages stimulated by LPS or TNF- 伪 under normoxic conditions had no significant change (P 0.05). After stimulation with LPS or TNF- 伪 for 10 h, the expression of HIF-1 伪 and VEGF protein in macrophages increased significantly, and the expression of HIF-1 伪 and VEGF protein in macrophages was significantly increased after SSZ pretreatment or L-NMMA treatment. The expression levels of HIF-1 伪 and VEGF protein in macrophages were significantly lower than those stimulated by LPS or TNF- 伪, and the expression levels of HIF-1 伪 and VEGF protein in the combination of SSZ and L-NMMA were significantly lower than those in the LPS or TNF- 伪 alone stimulated groups, but the expression levels of HIF-1 伪 and VEGF protein were significantly lower than those of LPS or TNF- 伪 alone, but the expression levels of HIF-1 伪 and VEGF protein were significantly lower than those of SSZ or L-NMMA alone. There was no significant difference in the level of HIF-1 伪 protein between time and time. The level of VEGF in P0.05 was significantly decreased by P0.05. Griess reaction showed that there was no significant difference in the level of HIF-1 伪 protein. Under the stimulation of LPS or TNF- 伪, the determination of nitrite in culture supernatant of normal control group was significantly higher than that of P0.05, L-NMMA or SSZ. The determination of nitrite in culture supernatant was significantly lower than that in LPS or TNF- 伪 alone. The levels of HIF-1 伪 and VEGF protein in macrophages treated with DETA-NO in normoxic environment for 4 h were significantly higher than those in normal control group (P 0.05). Conclusion NOS 伪 and TNF- 伪 can up-regulate the expression of HIF-1 伪 through post-transcriptional pathways such as NF- 魏 B and TNF- 伪, and the expression of HIF-1 伪 can further induce the expression of VEGF.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

【引证文献】