【摘要】： 氧化應(yīng)激在炎癥的發(fā)生發(fā)展過程中發(fā)揮著重要作用,一方面它可以直接損傷機體,另一方面它可以通過分子間相互作用間接地對機體造成損害。在參與炎癥過程的眾多復(fù)雜因子中,活性氧(ROS)和活性氮(RNS)非常重要,它們包括羥自由基、超氧陰離子、過氧化氫、一氧化氮、亞硝酸陰離子。有大量的實驗證明清除羥自由基和亞硝酸陰離子能夠明顯減輕炎癥的嚴重程度。最近的研究表明,氫氣能夠選擇性地清除羥自由基和亞硝酸陰離子。因此,氫氣很有可能通過這一途徑而具有抗炎的效應(yīng)。但是,通過呼吸道吸入氫氣的方法很不方便而且存在安全隱患,于是我們將氫氣通過高壓溶解于生理鹽水中,并探索這種含有氫氣的生理鹽水是否具有抗炎效應(yīng)。 本課題以角叉菜膠誘導小鼠足腫脹為實驗?zāi)Ｐ?研究氫生理鹽水對于該炎癥模型是否具有抗炎效應(yīng),并以巨噬細胞為切入點研究氫生理鹽水對炎性細胞的影響。 本實驗通過足爪容積測量儀檢測了小鼠足腫脹的程度;組織化學切片觀察了炎癥局部的中性粒細胞浸潤程度;酶聯(lián)免疫吸附試驗檢測了細胞培養(yǎng)上清中的TNF-α的含量;實時定量PCR檢測了RAW264.7及小鼠原代腹腔巨噬細胞中TNF-α的mRNA水平。 研究工作取得的主要結(jié)果如下: 1.氫生理鹽水能夠顯著抑制角叉菜膠誘導的足腫脹:以2.5ml/kg、5ml/kg、10ml/kg的氫生理鹽水的量處理角叉菜膠誘導足腫脹的動物模型,發(fā)現(xiàn)處理組腳腫脹的程度明顯低于對照組,而且5ml/kg這個劑量效果最好。 2.氫生理鹽水能夠顯著抑制炎癥局部中性粒細胞的浸潤:將氫生理鹽水處理組、非處理組及對照組的足爪做成石蠟切片,HE染色后觀察中性粒細胞浸潤程度,氫水處理組的中性粒細胞浸潤明顯比非處理組少。 3.氫生理鹽水能夠減少活化巨噬細胞的TNF-α的mRNA水平并能抑制巨噬細胞分泌TNF-α:氫生理鹽水和100ng/ml的LPS共同處理巨噬細胞1h,1h后裂解細胞通過實時定量PCR檢測TNF-α的mRNA水平,發(fā)現(xiàn)氫生理鹽水能夠抑制巨噬細胞TNF-α的mRNA的表達;氫生理鹽水和100ng/ml的LPS共同處理RAW264.7細胞4h,4h后收集上清檢測TNF-α,發(fā)現(xiàn)氫生理鹽水能夠顯著抑制由LPS引起的TNF-α產(chǎn)生,而且這種效應(yīng)具有劑量依賴性。 總之,氫生理鹽水不論在角叉菜膠誘導的足腫脹動物模型上還是在LPS激活的巨噬細胞上都具有明顯的抗炎效應(yīng),這一效應(yīng)很可能是通過清除羥自由基、亞硝酸陰離子等自由基來實現(xiàn)的。我們推測,氫生理鹽水憑借其有效性、安全性、方便性、廉價性,很可能成為我們以后治療某些炎癥疾病的藥物。在今后的工作中,我們將深入研究氫生理鹽水具有抗炎效應(yīng)的機制,如炎癥局部的自由基變化等。
[Abstract]:Oxidative stress plays an important role in the development of inflammation. On the one hand, it can directly damage the body, on the other hand, it can indirectly damage the body through intermolecular interaction. Among the many complex factors involved in inflammatory process, reactive oxygen species (ROS) and active nitrogen (RNS) are very important. They include hydroxyl radical, superoxide anion, hydrogen peroxide, nitric oxide, nitrite anion. A large number of experiments have shown that scavenging hydroxyl radicals and nitrite anions can significantly reduce the severity of inflammation. Recent studies have shown that hydrogen can selectively scavenge hydroxyl radicals and nitrite anions. Therefore, hydrogen may have an anti-inflammatory effect through this pathway. However, the method of inhaling hydrogen through respiratory tract is very inconvenient and has some potential safety problems. Therefore, we dissolve hydrogen into saline by high pressure, and explore whether the hydrogen containing saline has anti-inflammatory effect. In this study, the mouse foot swelling induced by carrageenin was used as the experimental model to study whether hydrophysiological saline has anti-inflammatory effect on the inflammatory model, and to study the effect of hydrogen physiological saline on inflammatory cells from macrophages. In this experiment, the degree of mouse foot swelling was detected by paw volume measuring instrument, the degree of neutrophil infiltration in inflammatory region was observed by histochemical section, the content of TNF- 偽 in supernatant of cell culture was detected by enzyme-linked immunosorbent assay (Elisa). The mRNA levels of TNF- 偽 in RAW264.7 and mouse primary peritoneal macrophages were measured by real time quantitative PCR. The main results of the research are as follows: 1. Hydrophysiological saline significantly inhibited paw swelling induced by carrageenin: the animal model of foot swelling was induced by carrageenin with 2.5 ml / kg of 5 ml / kg / kg of hydrophysiological saline and 10 ml / kg of hydrophysiological saline. It was found that the degree of foot swelling in the treatment group was significantly lower than that in the control group, and 5ml/kg was the best dose. 2. Hydrophysiological saline could significantly inhibit the infiltration of neutrophils in the inflammatory region. The paws of the hydrophysiological saline treated group, untreated group and control group were made into paraffin sections. The degree of neutrophil infiltration was observed after HE staining. The neutrophil infiltration in hydrogen water treatment group was significantly less than that in non-treated group. 3. HNS could reduce the mRNA level of TNF- 偽 in activated macrophages and inhibit the secretion of TNF- 偽 by macrophages: HNS and 100ng/ml LPS co-treated macrophages for 1 h. After 1 hour, the mRNA level of TNF- 偽 was detected by real-time quantitative PCR. It was found that hydrophysiological saline could inhibit the mRNA expression of TNF- 偽 in macrophages. The supernatant of RAW264.7 cells was collected for 4 h after co-treatment of HNS and LPS of 100ng/ml for 4 h. It was found that HNS could significantly inhibit the production of TNF- 偽 induced by LPS, and this effect was dose-dependent. In conclusion, both in carrageenin-induced foot swelling animal models and in LPS activated macrophages, hydrogen saline has a significant anti-inflammatory effect, probably by scavenging hydroxyl radicals. Nitrite anion and other free radicals to achieve. We speculate that hydrophysiological saline, because of its efficacy, safety, convenience and cheapness, is likely to become a drug for the treatment of certain inflammatory diseases in the future. In the future, we will further study the mechanism of anti-inflammatory effect of hydrophysiological saline, such as the changes of free radicals in inflammation.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R363

【引證文獻】

相關(guān)博士學位論文 前1條

1 以敏;桃仁改善不同病因所致血液循環(huán)障礙的藥效及相關(guān)分子機制研究[D];廣西醫(yī)科大學;2012年

相關(guān)碩士學位論文 前1條

1 楊麗珍;不同制備工藝葛根芩連湯抗感染作用的比較研究[D];黑龍江中醫(yī)藥大學;2011年

，

本文編號：2322116