LPS诱导的急性炎症中HIF-1α表达增加的机制研究

发布时间：2018-03-09 04:17

本文选题：HIF-1α　切入点：VEGF　出处：《苏州大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:从体内和体外两方面,探讨NF-κB抑制剂N-乙酰半胱氨酸(NAC)对脂多糖(LPS)诱导的小鼠急性炎症组织中和体外培养的小鼠巨噬细胞内HIF-1表达的影响及其意义。方法:将Balb/c小鼠分为正常对照组、炎症组、炎症用药组。炎症组采用卡介苗(BCG)配合脂多糖(LPS)尾静脉注射法建立小鼠急性全身性炎症损伤模型。炎症用药组在尾静脉注射脂多糖(LPS)前30分钟腹腔注射NAC,正常对照组给予等量PBS。于注射后5h立即摘除小鼠眼球放血、脱臼法处死小鼠,取出肝脏和肺脏进行常规石蜡切片、HE染色观察脏器的病理改变,免疫组织化学SP法观察HIF-1α及VEGF在组织中的表达,采用RT-PCR法半定量检测组织中HIF-1、VEGF基因mRNA的表达水平,应用ELISA法检测血清TNF-α的表达水平。另取正常Balb/c小鼠,分离腹腔巨噬细胞并将其分为正常对照组、LPS刺激组、不同剂量NAC+LPS组,2小时后将LPS溶液加入LPS组和相应浓度的NAC+LPS组使其终浓度达0.8ug/ml,正常对照组给予等量PBS。培养10小时后,采用RT-PCR法半定量检测各组HIF-1α、TNF-α基因mRNA的表达水平,应用细胞免疫化学染色法检测巨噬细胞HIF-1α、VEGF蛋白的表达水平。结果:体内实验中,炎症组小鼠血清TNF-α蛋白水平较正常对照组显著增加(P 0.01)。炎症用药组小鼠血清TNF-α蛋白水平较炎症组显著降低(P 0.01),肝及肺组织HIF-1α基因表达在mRNA的水平无显著差异(P0.05),VEGF基因表达的mRNA显著降低(P 0.01),HIF-1α、VEGF在蛋白水平上显著降低(P 0.01)。体外培养中,与LPS刺激组比较,NAC+LPS组巨噬细胞HIF-1α基因表达的mRNA无明显改变、TNF-α基因表达的mRNA明显减少。HIF-1α、VEGF蛋白表达显著减少(P 0.01)。结论:预先给予NAC,可使LPS诱导的急性全身炎症反应小鼠血清TNF-α水平显著降低的同时,肝和肺组织HIF-1α蛋白表达水平、VEGF基因mRNA与蛋白表达水平均显著降低;体外常氧条件下培养时,加入NAC可降低LPS刺激下小鼠巨噬细胞TNF-α的基因表达,TNF-α水平的降低可能通过HIF-1α转录后途径下调该蛋白在细胞内的水平,进而下调靶基因VEGF的表达。提示在急性炎症反应时,NF-κB信号通路通过增加TNF-α的表达在转录后水平影响HIF-1α的表达水平。
[Abstract]:Aim: to investigate the effect of NF- 魏 B inhibitor N-acetylcysteine on the expression of HIF-1 in murine acute inflammatory tissues induced by lipopolysaccharide (LPS) and in vitro cultured mouse macrophages. Methods: Balb/c mice were divided into normal control group and inflammatory group. Inflammatory drug group. The inflammatory group was injected with BCG (BCG) combined with lipopolysaccharide (LPS) into tail vein to establish acute systemic inflammatory injury model in mice. The inflammatory drug group was injected intraperitoneally with NAC30 minutes before injection of lipopolysaccharide LPS30 minutes before injection of lipopolysaccharide. The mice in the irradiation group were given the same amount of PBS. the eyeballs were extirpated at 5 hours after injection. The mice were killed by dislocated method, the liver and lungs were taken out for routine paraffin sections and HE staining to observe the pathological changes of the organs, and the expression of HIF-1 伪 and VEGF in the tissues were observed by immunohistochemical SP method. RT-PCR method was used to detect the expression of HIF-1 mRNA mRNA in tissues, ELISA method was used to detect the expression level of TNF- 伪 in serum, and peritoneal macrophages were isolated from normal Balb/c mice and divided into normal control group and LPS-stimulated group. LPS solution was added to LPS group and NAC LPS group for 2 hours to make the final concentration reach 0.8ugmml. the normal control group was given the same amount of PBSs. After 10 hours of culture, the mRNA expression of HIF-1 伪 TNF- 伪 gene was detected by semi-quantitative RT-PCR assay in each group. The expression of HIF-1 伪 -VEGF protein in macrophages was detected by immunocytochemical staining. Results: in vivo experiments, Serum TNF- 伪 protein level in inflammatory group was significantly higher than that in normal control group (P 0.01), serum TNF- 伪 protein level in inflammatory medication group was significantly lower than that in inflammatory group (P 0.01), and the expression of HIF-1 伪 gene in liver and lung tissues had no significant difference in mRNA level. The expression of mRNA significantly decreased the protein level of HIF-1 伪. Compared with LPS stimulation group, the expression of HIF-1 伪 gene in macrophages was not significantly changed by mRNA in NAC LPS group. The expression of mRNA in TNF- 伪 gene was significantly decreased. HIF-1 伪 -VEGF protein expression was significantly decreased in NAC LPS group than that in NAC LPS group (P 0.01). Conclusion: the serum TNF- 伪 level of acute systemic inflammatory reaction mice induced by LPS was significantly decreased by pretreatment with NAC- 伪, while the expression level of HIF-1 伪 protein in liver and lung tissues and the mRNA and protein expression levels of HIF-1 gene in liver and lung tissues were significantly decreased. When cultured under normoxic conditions in vitro, the addition of NAC could reduce the expression of TNF- 伪 gene in mouse macrophages stimulated by LPS. The decrease of TNF- 伪 gene expression may down-regulate the intracellular level of TNF- 伪 through HIF-1 伪 posttranscriptional pathway. Then down-regulated the expression of target gene VEGF, suggesting that NF- 魏 B signaling pathway affects the expression of HIF-1 伪 at post-transcriptional level by increasing the expression of TNF- 伪 in acute inflammatory response.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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