本文選題：氯化鑭(LaCl_3)　切入點：脂多糖(LPS　出處：《南昌大學》2007年碩士論文

【摘要】： 目的和意義: 一氧化氮(nitric oxide,NO)是炎癥反應過程中的重要介質(zhì)和調(diào)節(jié)因子,它雖可殺滅侵入機體的病原微生物,維持機體正常的免疫防御功能,但過量NO也對宿主細胞產(chǎn)生損傷作用。體內(nèi)NO生成的唯一途徑是由一氧化氮合酶(inducible nitric oxide synthase,iNOS)催化L-精氨酸合成。研究證明,脂多糖(lipopolysaccharide,LPS)等刺激因子能誘導單核巨噬細胞高表達iNOS,從而合成過量NO,引起一系列嚴重病理反應,如感染性休克和多器官功能衰竭(multiple organ failure,MOF)等。因此抑制iNOS的過量表達以控制NO的產(chǎn)生,對臨床相關(guān)疾病的治療具有重要意義。鑭是一種具有多種生物學活性的稀土元素,我們的前期研究已證實鑭具有結(jié)合LPS抑制LPS生物學效應的作用,本研究進一步探討鑭對LPS誘導的小鼠巨噬細胞產(chǎn)生NO的影響,同時觀察鑭對iNOS基因轉(zhuǎn)錄的重要調(diào)控因子-核轉(zhuǎn)錄因子-κB (nuclear factor-kappaB,NF-κB)活化的可能影響,以探討鑭影響iNOS表達的可能機制,為研究NO拮抗劑及開發(fā)稀土的藥用價值提供實驗依據(jù)。 研究內(nèi)容和方法: 1.通過MTT法觀察不同濃度氯化鑭(Lanthanum chloride ,LaCl_3)(2.5μmol/L～40μmol/L)對小鼠巨噬細胞生長活性的影響。 2.采用免疫熒光細胞化學染色、Western Blotting及RT-PCR方法觀察LaCl_3對LPS誘導的小鼠巨噬細胞iNOS蛋白及基因表達的影響,同時觀察NO產(chǎn)物的變化。 3.采用免疫熒光細胞化學染色及Western Blotting方法觀察LaCl_3對LPS活化小鼠巨噬細胞NF-κB的影響。 結(jié)果: 1.一定濃度的LaCl_3(2.5～40μmol/L)對小鼠巨噬細胞的生長活性無明顯影響。 2.免疫熒光細胞化學染色及Western Blotting結(jié)果顯示低濃度的LaCl_3(2.5μmol/L)能顯著抑制LPS誘導小鼠巨噬細胞iNOS的蛋白表達,而對正常培養(yǎng)的小鼠巨噬細胞iNOS蛋白表達無明顯影響。 3. RT-PCR結(jié)果顯示LaCl_3能顯著抑制LPS誘導小鼠巨噬細胞iNOSmRNA的表達,而對正常培養(yǎng)的小鼠巨噬細胞iNOSmRNA表達無明顯影響。 4. NO含量測定結(jié)果顯示LaCl_3能顯著抑制LPS誘導小鼠巨噬細胞產(chǎn)生NO。 5. NF-κB /p65活化狀況檢測結(jié)果顯示,LaCl_3能顯著抑制LPS誘導小鼠巨噬細胞NF-κB /p65核轉(zhuǎn)位,而對正常培養(yǎng)的小鼠巨噬細胞NF-κB /p65活性無明顯影響。 結(jié)論: 1.LaCl_3能顯著抑制LPS誘導小鼠巨噬細胞iNOS基因及蛋白的表達,從而顯著降低NO的分泌水平。 2.LaCl_3可能通過抑制LPS誘導小鼠巨噬細胞NF-κB /p65的活化,從而抑制iNOS的表達及NO的產(chǎn)生。
[Abstract]:Purpose and significance:Nitric oxide nitric oxide (no) is an important mediator and regulatory factor in the process of inflammatory reaction. Although it can kill the pathogenic microorganisms that invade the body and maintain the normal immune defense function of the body, excessive no also damages the host cells.The only way to produce no in vivo is to catalyze the synthesis of L-arginine by inducible nitric oxide synthase iNOS.It has been proved that lipopolysaccharide and other stimulating factors can induce the high expression of iNOS in mononuclear macrophages, and induce a series of severe pathological reactions, such as septic shock and multiple organ failure.Therefore, inhibiting the overexpression of iNOS in order to control the production of no is of great significance for the treatment of clinically-related diseases.Lanthanum is a rare earth element with many biological activities. Our previous studies have confirmed that lanthanum can inhibit the biological effects of LPS combined with LPS. In this study, we further investigated the effect of lanthanum on the production of no by mouse macrophages induced by LPS.At the same time, the possible effects of lanthanum on the activation of nuclear factor- 魏 B nuclear factor-kappa B, an important regulatory factor of iNOS gene transcription, were observed to explore the possible mechanism of lanthanum affecting the expression of iNOS, and to provide experimental evidence for studying no antagonists and exploiting the medicinal value of rare earths.Contents and methods of the study:1.The effects of Lanthanum chloride (2.5 渭 mol/L~40 渭 mol / L) at different concentrations on the growth activity of mouse macrophages were studied by MTT method.2.The effects of LaCl_3 on the expression of iNOS protein and gene in mouse macrophages induced by LPS were observed by immunofluorescence cytochemical staining and RT-PCR. The changes of no products were also observed.3.Immunofluorescence cytochemical staining and Western Blotting were used to observe the effect of LaCl_3 on NF- 魏 B in LPS activated mouse macrophages.Results:1.A certain concentration of LaCl_3(2.5~40 渭 mol / L had no effect on the growth activity of mouse macrophages.2.The results of immunofluorescence cytochemistry and Western Blotting showed that low concentration of LaCl_3(2.5 渭 mol / L significantly inhibited the expression of iNOS protein in murine macrophages induced by LPS, but had no effect on the expression of iNOS protein in murine macrophages cultured in normal culture.3.RT-PCR results showed that LaCl_3 could significantly inhibit the expression of iNOSmRNA in murine macrophages induced by LPS, but had no effect on the expression of iNOSmRNA in normal cultured mouse macrophages.4.The results showed that LaCl_3 could significantly inhibit the production of no in mouse macrophages induced by LPS.5.The results of activation of NF- 魏 B / p65 showed that LaCl3 could significantly inhibit the nuclear translocation of mouse macrophage NF- 魏 B / p65 induced by LPS, but had no effect on the activity of NF- 魏 B / p65 in normal cultured mouse macrophages.Conclusion:1.LaCl_3 significantly inhibited the expression of iNOS gene and protein in murine macrophages induced by LPS, thus significantly reduced the level of no secretion.2.LaCl_3 may inhibit the expression of iNOS and the production of no by inhibiting the activation of NF- 魏 B / p65 in mouse macrophages induced by LPS.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R363

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