發(fā)布時(shí)間：2018-10-08 06:34

【摘要】：急性肺損傷（Acute Lung Injury，ALI）是臨床上比較常見的一種危重病癥，它是過度的炎性反應(yīng)在肺部的表現(xiàn)。因其發(fā)病急且嚴(yán)重，發(fā)病率和死亡率高，到目前為止仍無有效手段和特效藥治療。當(dāng)機(jī)體發(fā)生急性肺損傷時(shí),會出現(xiàn)諸多的病理變化，其中最為顯著地是：肺內(nèi)過度失控的炎性反應(yīng)，肺泡表面活性物質(zhì)的降低、肺水腫和肺膨脹不全，呼吸肌疲勞導(dǎo)致通氣不足而產(chǎn)生的二氧化碳過多蓄積等癥狀。如果急性肺損傷得不到及時(shí)的治療，隨著病程的發(fā)展，其有可能會發(fā)展成為急性呼吸窘迫綜合征，而使疾病無法控制，最終會導(dǎo)致死亡。據(jù)報(bào)道，急性肺損傷的發(fā)病率逐年升高,其死亡率甚至高達(dá)40%~60%。脂多糖（Lipopolysaccharide，LPS）是炎癥反應(yīng)經(jīng)典的誘導(dǎo)劑，作為革蘭氏陰性菌細(xì)胞壁上的主要成分，是細(xì)菌治病的重要毒素，也是引起急性肺損傷的重要原因之一。當(dāng)機(jī)體受到革蘭氏陰性菌感染時(shí)，LPS會與細(xì)胞膜上的受體結(jié)合激活胞內(nèi)一系列的信號傳導(dǎo)途徑，致使上皮細(xì)胞、內(nèi)皮細(xì)胞、單核/巨噬細(xì)胞等合成和釋放大量的內(nèi)源性生物活性因子如炎癥細(xì)胞因子、趨化因子、生長因子等，這些生物活性因子可以通過血液循環(huán)到達(dá)全身各處，引起全身性的炎癥反應(yīng)，由此會引起中毒性休克、全身炎癥反應(yīng)綜合癥和多器官功能障礙綜合癥等惡性疾病。 積雪草(Centella Asiatsca)作為一種傳統(tǒng)中藥材，用藥歷史悠久,最早記載于《神農(nóng)本草經(jīng)》，性寒，味苦、辛，，具有清熱利濕、解毒消腫之功效，近代研究發(fā)現(xiàn)積雪草具有抗氧化，抵抗炎癥，抑制腫瘤細(xì)胞生長等作用。本研究通過LPS誘導(dǎo)RAW264.7細(xì)胞的炎癥反應(yīng)和Balb/c小鼠的急性肺損傷模型，從體外和體內(nèi)兩個(gè)方面說明了積雪草粗提取物對炎癥反應(yīng)有比較明顯的抑制作用，從而發(fā)現(xiàn)了其對于小鼠急性肺損傷的各種病變有顯著地緩解作用，一定程度上起到保護(hù)小鼠肺組織的作用，初步探討了積雪草對于LPS引發(fā)的炎癥反應(yīng)會產(chǎn)生抑制作用的分子機(jī)制。 實(shí)驗(yàn)方法：利用LPS滴鼻建立小鼠急性肺損傷模型，并且利用酶聯(lián)免疫吸附試驗(yàn)（Enzyme-linkedimmunosorbent assay，ELISA）檢測了腫瘤壞死因子-α（tumornecrosis factor-α，TNF-α）和白細(xì)胞介素-6（IL-6）在細(xì)胞上清和小鼠肺泡灌洗液中的含量。同時(shí)，免疫蛋白印跡（Western blot）實(shí)驗(yàn)檢測NF-κB信號通路的活化，我們還觀察了小鼠肺組織病理變化，測定了肺組織中髓過氧化物酶(MPO)含量及肺濕/干重比(W/D)。 實(shí)驗(yàn)結(jié)果：在LPS誘導(dǎo)的炎癥反應(yīng)中，積雪草粗提取物能顯著的減少炎性細(xì)胞因子TNF-α和IL-6的表達(dá)水平，并且呈現(xiàn)一定劑量依賴性，高劑量效果顯著。另外，通過觀察小鼠肺部病理組織變化，測定小鼠肺組織內(nèi)髓過氧化物酶（Myeloperoxidase，MPO）以及干濕重比等指標(biāo)，綜合的說明了積雪草粗提取物對于小鼠急性肺損傷的諸多病變有顯著地緩解，表現(xiàn)出了一定程度的治療作用。并且Western blot實(shí)驗(yàn)結(jié)果說明了積雪草粗提取物顯著的減弱了NF-κB信號通路的活化，為其機(jī)制的研究奠定基礎(chǔ)。 實(shí)驗(yàn)結(jié)論：基于以上的研究，我們證明了積雪草具有體內(nèi)外的抗炎效果，并且對于小鼠急性肺損傷病變的緩解效果顯著，為該藥的廣泛使用提供了理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。
[Abstract]:Acute lung injury (ALI) is a very common serious condition in clinic, which is an excessive inflammatory response in the lungs. Due to its acute and serious morbidity and mortality, there has been no effective means and therapeutic treatment so far. In the case of acute lung injury in the body, many pathological changes occur, most notably: inflammatory response of over-control in the lungs, decreased alveolar surfactant, pulmonary edema, and incomplete lung expansion, Respiratory muscle fatigue leads to excessive accumulation of carbon dioxide and other symptoms due to insufficient ventilation. If acute lung injury is not treated in a timely manner, as the course progresses, it may develop into acute respiratory distress syndrome, which will not control the disease and ultimately lead to death. It was reported that the incidence of acute lung injury increased year by year, and the mortality rate was even 40% ~ 60%. Lipopolysaccharide (LPS) is a classical inducer of inflammatory response. As the main component of the cell wall of gram-negative bacteria, it is one of the most important causes of acute lung injury. When the body is infected by Gram-negative bacteria, the LPS binds to the receptors on the cell membrane to activate a series of signaling pathways in the cell, such that epithelial cells, endothelial cells, monocytes/ macrophages, and the like are synthesized and released a large number of endogenous bioactive factors such as inflammatory cytokines, Chemokine, growth factor, etc., these biological activity factors can reach the whole body through the blood circulation, cause systemic inflammatory reaction, thus can cause toxic shock, systemic inflammatory response syndrome and multiple organ dysfunction syndrome and other malignant diseases. Centella Asiana sca, as a traditional Chinese medicinal material, has a long history, which is first recorded in Shennong Cao Jing>, cold, bitter and pungent, and has the effects of clearing away heat, promoting diuresis, removing toxic materials and detumescence, and modern research finds that the traditional Chinese medicine has antioxidant and resistance. Inflammation, inhibition of tumor cell growth, etc. In this study, the inflammatory response of RAW264.7 cells was induced by LPS and the model of acute lung injury in BALB/ c mice. It has been found that it has a significant effect on various pathological changes of acute lung injury in mice, which plays a role in protecting the lung tissues of mice, and preliminarily probes into the molecules which can inhibit the inflammatory response induced by LPS. Methods: The acute lung injury model of mice was established by LPS nasal drops, and tumor necrosis factor-7721 (TNF-hIL) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay (ELISA). The activity of NF-Sepharose B signaling pathway was detected by Western blot. The pathological changes of lung tissue were also observed. The content of MPO in lung tissue and the ratio of lung wet/ dry weight were determined. (W/ D). The results showed that in the LPS-induced inflammatory response, the crude extract could significantly reduce the expression level of IL-6 in inflammatory cytokines and present a dose-dependent dose. In addition, by observing the changes of lung pathological tissues of mice, the indexes such as myelopoxidase (MPO) and dry-wet weight ratio in lung tissues of mice were measured. There was a significant response to the lesion, showing a The results of Western blot showed that the crude extract significantly attenuated the activation of NF-Sepharose B signaling pathway, Conclusion: Based on the above researches, we have demonstrated that the anti-inflammatory effect in mice has an external anti-inflammatory effect, and has a significant effect on the acute lung injury in mice, and it is widely used in the study.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S853.7

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