本文選題：脂多糖 + 腫瘤壞死因子-α　； 參考：《蘇州大學(xué)》2009年碩士論文

【摘要】： 目的:研究脂多糖(LPS)和腫瘤壞死因子-α(TNF-α)對小鼠腹腔巨噬細(xì)胞缺氧誘導(dǎo)因子-1α(HIF-1α)表達(dá)的相關(guān)信號通路。 方法:自BALB/c小鼠腹腔分離獲得巨噬細(xì)胞進(jìn)行體外培養(yǎng),觀察NF-κB抑制劑柳氮磺胺吡啶(SSZ)或一氧化氮合酶(NOS)抑制劑L-單甲基精氨酸(L-NMMA)或在兩者聯(lián)合作用對LPS或TNF-α誘導(dǎo)細(xì)胞部分信號分子表達(dá)的影響,以逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)法檢測培養(yǎng)細(xì)胞HIF-1αmRNA水平的變化,用免疫細(xì)胞化學(xué)染色法檢測培養(yǎng)細(xì)胞中HIF-1α和VEGF蛋白表達(dá)的變化,用Griess反應(yīng)檢測培養(yǎng)上清中亞硝酸鹽濃度的變化。 結(jié)果:常氧條件下,LPS或TNF-α刺激后,巨噬細(xì)胞的HIF-1αmRNA水平無明顯變化(P0.05)。巨噬細(xì)胞經(jīng)LPS或TNF-α刺激10h后,HIF-1α和VEGF蛋白表達(dá)水平明顯增加(P0.05);經(jīng)SSZ預(yù)處理或L-NMMA作用后,巨噬細(xì)胞HIF-1α和VEGF蛋白的表達(dá)水平較單純LPS或TNF-α刺激時明顯減少(P0.05);在SSZ和L-NMMA聯(lián)合作用下, HIF-1α和VEGF蛋白的表達(dá)水平明顯低于LPS或TNF-α單獨(dú)刺激組者(P0.05),但與SSZ或L-NMMA單獨(dú)使用時的抑制效應(yīng)相比,HIF-1α蛋白水平未見明顯差異(P0.05),VEGF蛋白水平明顯下降(P0.05)。Griess反應(yīng)顯示,正常對照組培養(yǎng)上清中亞硝酸鹽的測定值較低。LPS或TNF-α刺激下,培養(yǎng)上清中亞硝酸鹽的測定值明顯升高(P0.05),L-NMMA或SSZ作用后,培養(yǎng)上清中亞硝酸鹽的測定值較LPS或TNF-α單獨(dú)刺激時者明顯降低(P0.05)。常氧環(huán)境下,DETA-NO作用巨噬細(xì)胞4h后,HIF-1α和VEGF蛋白表達(dá)水平較正常對照組者明顯升高(P0.05)。 結(jié)論:LPS和TNF-α可通過NF-κB和NOS等轉(zhuǎn)錄后途徑上調(diào)HIF-1α的表達(dá), HIF-1α的表達(dá)可進(jìn)一步誘導(dǎo)其靶基因VEGF的表達(dá)。
[Abstract]:Aim: to investigate the signal pathways associated with the expression of hypoxia-inducible factor-1 偽 (HIF-1 偽) in mouse peritoneal macrophages by lipopolysaccharide (LPS) and tumor necrosis factor- 偽 (TNF- 偽). 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- 偽. The level of HIF-1 偽 mRNA was detected by reverse transcriptase polymerase chain reaction (RT PCR), the expression of HIF-1 偽 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 偽 mRNA in macrophages stimulated by LPS or TNF- 偽 under normoxic conditions had no significant change (P 0.05). After stimulation with LPS or TNF- 偽 for 10 h, the expression of HIF-1 偽 and VEGF protein in macrophages increased significantly, and the expression of HIF-1 偽 and VEGF protein in macrophages was significantly increased after SSZ pretreatment or L-NMMA treatment. The expression levels of HIF-1 偽 and VEGF protein in macrophages were significantly lower than those stimulated by LPS or TNF- 偽, and the expression levels of HIF-1 偽 and VEGF protein in the combination of SSZ and L-NMMA were significantly lower than those in the LPS or TNF- 偽 alone stimulated groups, but the expression levels of HIF-1 偽 and VEGF protein were significantly lower than those of LPS or TNF- 偽 alone, but the expression levels of HIF-1 偽 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 偽 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 偽 protein. Under the stimulation of LPS or TNF- 偽, 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- 偽 alone. The levels of HIF-1 偽 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 偽 and TNF- 偽 can up-regulate the expression of HIF-1 偽 through post-transcriptional pathways such as NF- 魏 B and TNF- 偽, and the expression of HIF-1 偽 can further induce the expression of VEGF.
【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】：2009
【分類號】：R363

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張文俊;牛膝多糖對脂多糖應(yīng)激斷奶仔豬生長性能、免疫功能和腸道功能的影響[D];湖南農(nóng)業(yè)大學(xué);2011年

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本文編號：1821288