本文選題：NaCl + LPS��； 參考：《濟(jì)南大學(xué)》2017年碩士論文

【摘要】：目的:研究高鹽對M1型巨噬細(xì)胞極化的調(diào)控,并在內(nèi)毒素休克炎癥疾病模型中得到驗證,這為臨床一些疾病的干預(yù)和治療提供了理論依據(jù),同時為我們下一步探究其機(jī)制打好堅實的基礎(chǔ)。方法:1.用GM-CSF誘導(dǎo)小鼠骨髓來源的原代巨噬細(xì)胞(BMDMs),在加入LPS(200 ng/ml)和IFN-γ(10 ng/ml)之前,用不同濃度的NaCl預(yù)處理BMDMs 2小時。(1)用CCK8檢測細(xì)胞活力的方法檢測不同濃度的NaCl對M1型巨噬細(xì)胞的活力的影響,確定一個合適的高鹽濃度用于后續(xù)的實驗。(2)用該合適的高鹽濃度預(yù)處理BMDMs,分別在處理后的6、12和24 h收取細(xì)胞培養(yǎng)上清,用ELISA檢測M1型巨噬細(xì)胞因子IL-6和IL-12p40的表達(dá)。(3)用不同濃度的Na Cl(5、10和20 mM)預(yù)處理BMDMs,24小時后,收取細(xì)胞并提取總RNA,RT-PCR檢測IL-6和IL-12p40在mRNA水平的表達(dá)。(4)流式細(xì)胞術(shù)檢測胞內(nèi)抗體IL-12p40的平均熒光強(qiáng)度。(5)流式細(xì)胞術(shù)檢測M1型巨噬細(xì)胞表面標(biāo)記CD86和MHCII的表達(dá)。(6)Western Blot檢測IRF5蛋白的表達(dá)。2.成功誘導(dǎo)BMDMs,將細(xì)胞分為兩組,其中一組用NaCl預(yù)處理6小時,另外一組細(xì)胞不做任何處理,收取細(xì)胞,使其濃度為1×107/ml。將20只老鼠分為兩組,每組10只,分別將200μl上述兩組細(xì)胞腹腔注射入小鼠體內(nèi)。(1)24小時后,每只小鼠腹腔注射800μg的LPS,觀察小鼠死亡率。(2)24小時后,每只小鼠腹腔注射200μg的LPS,12小時后小鼠眼球取血,斷頸處死小鼠,抽取腹腔細(xì)胞,獲得小鼠肺、肝臟和脾。(1)病理切片HE染色顯示小鼠肺和肝臟的損傷情況。(2)ELISA檢測小鼠血清中IL-6和IL-12p40的表達(dá)。(3)RT-PCR檢測小鼠腹腔和脾細(xì)胞中IL-6和IL-12p40在mRNA水平的表達(dá)。結(jié)果:1.(1)高濃度的NaCl(≥40 mM)減弱了M1型巨噬細(xì)胞的活力。合適的高鹽濃度為20 mM,該濃度不會隨著時間的增加影響細(xì)胞活力。(2)ELISA結(jié)果顯示,NaCl預(yù)處理后,IL-6和IL-12p40的表達(dá)顯著降低,并且具有時間和劑量的依賴性。(3)RT-PCR顯示,與對照組相比,NaCl預(yù)處理組IL-6和IL-12p40的表達(dá)明顯降低了,且具有劑量依賴性。(4)流式細(xì)胞術(shù)顯示,相對于正常組,NaCl預(yù)處理組IL-12p40的表達(dá)也顯著減少。(5)流式細(xì)胞術(shù)顯示,與正常組相比,NaCl預(yù)處理組M1型巨噬細(xì)胞表面標(biāo)記CD86和MHCII的表達(dá)并沒有明顯的變化。(6)Western Blot顯示,與對照組相比,NaCl預(yù)處理組IRF5的表達(dá)也顯著減少。2.(1)注射NaCl預(yù)處理的BMDMs組小鼠的死亡率明顯降低。(2)HE染色顯示,NaCl干預(yù)可明顯抑制LPS所致的肺和肝臟組織的損傷。(3)注射NaCl預(yù)處理的BMDMs小鼠體內(nèi),LPS所致的M1型巨噬細(xì)胞的極化明顯減少,其M1型巨噬細(xì)胞因子IL-6和IL-12p40的表達(dá)顯著減少。結(jié)論:1.NaCl可抑制體外M1型巨噬細(xì)胞的極化,其細(xì)胞因子IL-6和IL-12p40的表達(dá)顯著降低,其機(jī)制可能是NaCl減少了M1型巨噬細(xì)胞的轉(zhuǎn)錄因子IRF5的表達(dá)。2.在小鼠體內(nèi),NaCl減少了M1型巨噬細(xì)胞的極化,從而減輕了LPS所致的內(nèi)毒素?fù)p傷,減弱了LPS所引起的炎癥反應(yīng)。
[Abstract]:Objective: to study the effect of high salt on the polarization of M1 macrophages and to verify it in the model of inflammatory diseases of endotoxic shock, which provides a theoretical basis for the intervention and treatment of some diseases. At the same time for our next step to explore its mechanism to lay a solid foundation. Method 1: 1. Mouse bone marrow-derived primary macrophages were induced by GM-CSF. Before the addition of LPS200 ng / ml and IFN- 緯 10 ng / ml, BMDMs were pretreated with different concentrations of NaCl for 2 hours. 1) the effects of different concentrations of NaCl on the activity of M1 macrophages were detected by CCK8 method. To determine a suitable concentration of high salt for further experiment. (2) to pretreat BMDMswith the appropriate concentration of high salt, and to collect the supernatant of cell culture at 6h and 24h after treatment, respectively. The expressions of IL-6 and IL-12p40 in M1 macrophage were detected by Elisa. The expression of IL-6 and IL-12p40 at mRNA level was detected by RT-PCR. Flow cytometry was used to detect the average fluorescence intensity of intracellular antibody IL-12p40. Flow cytometry was used to detect the expression of CD86 and MHCII on the surface of M1 macrophages. Western blot was used to detect the expression of IRF5 protein. BMDMswere successfully induced and divided into two groups. One group was pretreated with NaCl for 6 hours, the other group received cells without any treatment, and the concentration of the cells was 1 脳 10 ~ (7) 路ml ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1). Twenty mice were divided into two groups, 10 rats in each group. After intraperitoneal injection of 200 渭 l cells into mice for 24 hours, each mouse was injected intraperitoneally with 800 渭 g LPSs. The mortality of mice was observed after 24 hours. Each mouse was injected intraperitoneally with 200 渭 g LPS for 12 hours. The mice were killed by amputation of their necks, the peritoneal cells were extracted, and the lungs of the mice were obtained. The expression of IL-6 and IL-12p40 in serum was detected by Elisa. RT-PCR was used to detect the mRNA expression of IL-6 and IL-12p40 in peritoneal and splenic cells of mice. Results the activity of M1 macrophages was weakened by high concentration of NaCl1 (鈮，

本文編號：2016302