本文選題：積雪草酸 + 銀屑病�。� 參考：《江蘇大學》2017年碩士論文

【摘要】：背景:積雪草酸是一種主要存在于傘形科植物積雪草中的烏蘇烷型五環(huán)三萜酸,其資源豐富,目前主要分布于我國長江流域以南地區(qū)�，F(xiàn)有研究表明,積雪草酸具有抗腫瘤、抗炎癥、抗氧化、抗抑郁等多種藥理活性。銀屑病是一種病理表現(xiàn)為角質(zhì)形成細胞(keratinocyte,KC)的過度增殖和炎癥細胞浸潤的慢性皮膚病。HaCaT細胞是自發(fā)永生化的人角化細胞系,已廣泛用于許多皮膚疾病的研究。本實驗擬探討積雪草酸對咪喹莫特誘導的小鼠銀屑病模型及其對角質(zhì)形成細胞的作用和機制。方法:建立咪喹莫特誘導小鼠銀屑病模型,考察積雪草酸對小鼠皮損組織的影響;測定血清中IL-17、IL-23、TNF-α的含量來檢測炎癥因子的釋放;檢測小鼠皮損組織中PCNA、Bax、Bcl-2蛋白的表達。建立TNF-α誘導HaCaT細胞增殖的細胞模型,考察積雪草酸抑制TNF-α誘導HaCaT細胞增殖的分子機制。以不同濃度積雪草酸作用于TNF-α誘導HaCaT細胞24 h,MTT法檢測細胞活性;流式細胞術(shù)檢測細胞周期;免疫熒光染色法檢測NF-κB的核轉(zhuǎn)位;Western blot法檢測細胞蛋白NF-κB、p-NF-κB的表達。研究積雪草酸對HaCaT細胞凋亡的影響:MTT法檢測細胞活性;DCFH-DA染色檢測細胞內(nèi)及線粒體ROS生成;JC-1染色檢測線粒體膜電位,熒光素酶法檢測ATP含量,分析線粒體功能。結(jié)果:通過對皮膚的外觀觀察以及組織HE染色發(fā)現(xiàn),積雪草酸對咪喹莫特誘導小鼠銀屑病模型皮膚的銀屑樣癥狀具有較好的改善效果。檢測小鼠血清中IL-17、IL-23、TNF-α的含量發(fā)現(xiàn),咪喹莫特造成小鼠銀屑病模型時,IL-17、IL-23、TNF-α含量顯著升高(P0.01),積雪草酸給藥處理后能夠明顯降低TNF-α的升高,但是對IL-17、IL-23無明顯影響。檢測小鼠皮損組織蛋白的表達水平發(fā)現(xiàn),積雪草酸處理后,細胞核抗原PCNA表達水平降低、細胞凋亡蛋白Bax表達水平升高、Bcl-2表達水平降低。細胞水平研究結(jié)果顯示,積雪草酸能夠顯著抑制TNF-α誘導的HaCaT細胞增殖,阻滯細胞周期于G2期。同時我們發(fā)現(xiàn)積雪草酸能夠抑制TNF-α誘導的NF-κB磷酸化,并阻止其轉(zhuǎn)位入核。此外,積雪草酸處理能夠誘導HaCaT細胞內(nèi)ROS生成增多,促使線粒體膜電位倒塌,抑制細胞內(nèi)ATP的生成,最終誘導角質(zhì)形成細胞凋亡。結(jié)論:積雪草酸具有良好改善小鼠銀屑病的作用,表現(xiàn)為抑制角質(zhì)形成細胞的增殖。在細胞水平,積雪草酸能夠抑制TNF-α誘導HaCaT細胞增殖并顯著能夠損傷HaCaT細胞的線粒體,誘導細胞發(fā)生凋亡。其機制的可能與積雪草酸能夠抑制TNF-α誘導的NF-κB通路活化相關(guān)。
[Abstract]:Background: Asiaticoic acid is a kind of ursolane-type triterpenoid acid which mainly exists in the umbellidae plants. It is rich in resources and is mainly distributed in the south of the Yangtze River basin in China.Current studies have shown that oxalic acid has many pharmacological activities, such as anti-tumor, anti-inflammatory, anti-oxidation, anti-depressant and so on.Psoriasis is a chronic dermatosis, characterized by excessive proliferation of keratinocytes and infiltration of inflammatory cells in keratinocytes. HaCaT cells are spontaneous immortalized human keratinized cell lines, which have been widely used in the study of many skin diseases.The aim of this study was to investigate the effect and mechanism of oxalic acid on the psoriasis model induced by Imiquimote in mice and on keratinocytes.Methods: the model of psoriasis induced by imiquimod was established, the effect of oxalic acid on the skin lesions of mice was investigated, the levels of IL-17, IL-23 and TNF- 偽 in serum were determined to detect the release of inflammatory factors, and the expression of PCNA Baxax-Bcl 2 protein in the lesions of mice was detected.To establish a cell model of HaCaT cell proliferation induced by TNF- 偽, and to investigate the molecular mechanism of cytosolic oxalic acid inhibiting TNF- 偽 -induced proliferation of HaCaT cells.TNF- 偽 -induced HaCaT cells were treated with different concentrations of oxalic acid for 24 h. Flow cytometry was used to detect the cell cycle, and immunofluorescence staining was used to detect the nuclear translocation of NF- 魏 B and the expression of NF- 魏 B p-NF- 魏 B protein.The effect of oxalic acid on apoptosis of HaCaT cells; DCFH-DA staining was used to detect cell viability and mitochondrial ROS production; JC-1 staining was used to detect mitochondrial membrane potential; luciferase method was used to detect ATP content and mitochondrial function.Results: through the observation of skin appearance and tissue HE staining, it was found that snowy oxalic acid had a better effect on the silver-like symptoms of psoriasis model mice induced by Imiquimod.The content of IL-17, IL-23, TNF- 偽 in serum of mice was detected. It was found that the content of IL-17, IL-23, TNF- 偽 in mice psoriasis model was significantly increased when imiquimod was induced, but the increase of TNF- 偽 was significantly decreased after the administration of arachalic acid, but there was no significant effect on IL-17 IL-23.It was found that the expression of nuclear antigen (PCNA) was decreased and the expression of apoptotic protein (Bax) was increased and Bcl-2 was decreased after treated with Asiatidic acid.The results of cell-level study showed that cytosolic oxalic acid could significantly inhibit the proliferation of HaCaT cells induced by TNF- 偽 and arrest the cell cycle in G2 phase.At the same time, we found that oxalic acid can inhibit the phosphorylation of NF- 魏 B induced by TNF- 偽 and prevent its translocation into the nucleus.In addition, Asiaticoic acid could induce the increase of ROS production in HaCaT cells, induce the collapse of mitochondrial membrane potential, inhibit the formation of intracellular ATP, and eventually induce keratinocyte apoptosis.Conclusion: oxalic acid can improve psoriasis in mice and inhibit the proliferation of keratinocytes.At the cellular level, oxalic acid could inhibit the proliferation of HaCaT cells induced by TNF- 偽, significantly damage the mitochondria of HaCaT cells and induce apoptosis.The mechanism may be related to the inhibition of the activation of NF- 魏 B pathway induced by TNF- 偽 by oxalic acid.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5

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