镧抑制脂多糖诱导小鼠巨噬细胞产生一氧化氮的机制

发布时间：2018-04-02 21:17

本文选题：氯化镧(LaCl_3)　切入点：脂多糖(LPS　出处：《南昌大学》2007年硕士论文

【摘要】： 目的和意义: 一氧化氮(nitric oxide,NO)是炎症反应过程中的重要介质和调节因子,它虽可杀灭侵入机体的病原微生物,维持机体正常的免疫防御功能,但过量NO也对宿主细胞产生损伤作用。体内NO生成的唯一途径是由一氧化氮合酶(inducible nitric oxide synthase,iNOS)催化L-精氨酸合成。研究证明,脂多糖(lipopolysaccharide,LPS)等刺激因子能诱导单核巨噬细胞高表达iNOS,从而合成过量NO,引起一系列严重病理反应,如感染性休克和多器官功能衰竭(multiple organ failure,MOF)等。因此抑制iNOS的过量表达以控制NO的产生,对临床相关疾病的治疗具有重要意义。镧是一种具有多种生物学活性的稀土元素,我们的前期研究已证实镧具有结合LPS抑制LPS生物学效应的作用,本研究进一步探讨镧对LPS诱导的小鼠巨噬细胞产生NO的影响,同时观察镧对iNOS基因转录的重要调控因子-核转录因子-κB (nuclear factor-kappaB,NF-κB)活化的可能影响,以探讨镧影响iNOS表达的可能机制,为研究NO拮抗剂及开发稀土的药用价值提供实验依据。研究内容和方法: 1.通过MTT法观察不同浓度氯化镧(Lanthanum chloride ,LaCl_3)(2.5μmol/L～40μmol/L)对小鼠巨噬细胞生长活性的影响。 2.采用免疫荧光细胞化学染色、Western Blotting及RT-PCR方法观察LaCl_3对LPS诱导的小鼠巨噬细胞iNOS蛋白及基因表达的影响,同时观察NO产物的变化。 3.采用免疫荧光细胞化学染色及Western Blotting方法观察LaCl_3对LPS活化小鼠巨噬细胞NF-κB的影响。结果: 1.一定浓度的LaCl_3(2.5～40μmol/L)对小鼠巨噬细胞的生长活性无明显影响。 2.免疫荧光细胞化学染色及Western Blotting结果显示低浓度的LaCl_3(2.5μmol/L)能显著抑制LPS诱导小鼠巨噬细胞iNOS的蛋白表达,而对正常培养的小鼠巨噬细胞iNOS蛋白表达无明显影响。 3. RT-PCR结果显示LaCl_3能显著抑制LPS诱导小鼠巨噬细胞iNOSmRNA的表达,而对正常培养的小鼠巨噬细胞iNOSmRNA表达无明显影响。 4. NO含量测定结果显示LaCl_3能显著抑制LPS诱导小鼠巨噬细胞产生NO。 5. NF-κB /p65活化状况检测结果显示,LaCl_3能显著抑制LPS诱导小鼠巨噬细胞NF-κB /p65核转位,而对正常培养的小鼠巨噬细胞NF-κB /p65活性无明显影响。结论: 1.LaCl_3能显著抑制LPS诱导小鼠巨噬细胞iNOS基因及蛋白的表达,从而显著降低NO的分泌水平。 2.LaCl_3可能通过抑制LPS诱导小鼠巨噬细胞NF-κB /p65的活化,从而抑制iNOS的表达及NO的产生。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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