本文選題：HIF-1α　切入點：VEGF　出處：《蘇州大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:從體內(nèi)和體外兩方面,探討NF-κB抑制劑N-乙酰半胱氨酸(NAC)對脂多糖(LPS)誘導(dǎo)的小鼠急性炎癥組織中和體外培養(yǎng)的小鼠巨噬細(xì)胞內(nèi)HIF-1表達(dá)的影響及其意義。 方法:將Balb/c小鼠分為正常對照組、炎癥組、炎癥用藥組。炎癥組采用卡介苗(BCG)配合脂多糖(LPS)尾靜脈注射法建立小鼠急性全身性炎癥損傷模型。炎癥用藥組在尾靜脈注射脂多糖(LPS)前30分鐘腹腔注射NAC,正常對照組給予等量PBS。于注射后5h立即摘除小鼠眼球放血、脫臼法處死小鼠,取出肝臟和肺臟進(jìn)行常規(guī)石蠟切片、HE染色觀察臟器的病理改變,免疫組織化學(xué)SP法觀察HIF-1α及VEGF在組織中的表達(dá),采用RT-PCR法半定量檢測組織中HIF-1、VEGF基因mRNA的表達(dá)水平,應(yīng)用ELISA法檢測血清TNF-α的表達(dá)水平。另取正常Balb/c小鼠,分離腹腔巨噬細(xì)胞并將其分為正常對照組、LPS刺激組、不同劑量NAC+LPS組,2小時后將LPS溶液加入LPS組和相應(yīng)濃度的NAC+LPS組使其終濃度達(dá)0.8ug/ml,正常對照組給予等量PBS。培養(yǎng)10小時后,采用RT-PCR法半定量檢測各組HIF-1α、TNF-α基因mRNA的表達(dá)水平,應(yīng)用細(xì)胞免疫化學(xué)染色法檢測巨噬細(xì)胞HIF-1α、VEGF蛋白的表達(dá)水平。 結(jié)果:體內(nèi)實驗中,炎癥組小鼠血清TNF-α蛋白水平較正常對照組顯著增加(P 0.01)。炎癥用藥組小鼠血清TNF-α蛋白水平較炎癥組顯著降低(P 0.01),肝及肺組織HIF-1α基因表達(dá)在mRNA的水平無顯著差異(P0.05),VEGF基因表達(dá)的mRNA顯著降低(P 0.01),HIF-1α、VEGF在蛋白水平上顯著降低(P 0.01)。體外培養(yǎng)中,與LPS刺激組比較,NAC+LPS組巨噬細(xì)胞HIF-1α基因表達(dá)的mRNA無明顯改變、TNF-α基因表達(dá)的mRNA明顯減少。HIF-1α、VEGF蛋白表達(dá)顯著減少(P 0.01)。 結(jié)論:預(yù)先給予NAC,可使LPS誘導(dǎo)的急性全身炎癥反應(yīng)小鼠血清TNF-α水平顯著降低的同時,肝和肺組織HIF-1α蛋白表達(dá)水平、VEGF基因mRNA與蛋白表達(dá)水平均顯著降低;體外常氧條件下培養(yǎng)時,加入NAC可降低LPS刺激下小鼠巨噬細(xì)胞TNF-α的基因表達(dá),TNF-α水平的降低可能通過HIF-1α轉(zhuǎn)錄后途徑下調(diào)該蛋白在細(xì)胞內(nèi)的水平,進(jìn)而下調(diào)靶基因VEGF的表達(dá)。提示在急性炎癥反應(yīng)時,NF-κB信號通路通過增加TNF-α的表達(dá)在轉(zhuǎn)錄后水平影響HIF-1α的表達(dá)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 偽 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- 偽 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 偽 TNF- 偽 gene was detected by semi-quantitative RT-PCR assay in each group. The expression of HIF-1 偽 -VEGF protein in macrophages was detected by immunocytochemical staining. Results: in vivo experiments, Serum TNF- 偽 protein level in inflammatory group was significantly higher than that in normal control group (P 0.01), serum TNF- 偽 protein level in inflammatory medication group was significantly lower than that in inflammatory group (P 0.01), and the expression of HIF-1 偽 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 偽. Compared with LPS stimulation group, the expression of HIF-1 偽 gene in macrophages was not significantly changed by mRNA in NAC LPS group. The expression of mRNA in TNF- 偽 gene was significantly decreased. HIF-1 偽 -VEGF protein expression was significantly decreased in NAC LPS group than that in NAC LPS group (P 0.01). Conclusion: the serum TNF- 偽 level of acute systemic inflammatory reaction mice induced by LPS was significantly decreased by pretreatment with NAC- 偽, while the expression level of HIF-1 偽 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- 偽 gene in mouse macrophages stimulated by LPS. The decrease of TNF- 偽 gene expression may down-regulate the intracellular level of TNF- 偽 through HIF-1 偽 posttranscriptional pathway. Then down-regulated the expression of target gene VEGF, suggesting that NF- 魏 B signaling pathway affects the expression of HIF-1 偽 at post-transcriptional level by increasing the expression of TNF- 偽 in acute inflammatory response.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R363

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本文編號：1586951