【摘要】：目的：巨噬細(xì)胞吞噬氧化低密度脂蛋白(oxygenized low density lipoproteins, ox-LDL)并轉(zhuǎn)化為泡沫細(xì)胞。研究發(fā)現(xiàn),泡沫細(xì)胞大量死亡可導(dǎo)致局部堆積大量脂質(zhì),斑塊進(jìn)展甚至破裂。細(xì)胞死亡方式包括：自噬性死亡,凋亡和壞死。丙酮酸乙酯(Ethyl pyruvate,EP)是一種丙酮酸衍生物,多個(gè)研究顯示該藥物能通過抑制細(xì)胞自噬和凋亡,減少細(xì)胞死亡,從而發(fā)揮對臟器的保護(hù)作用。本研究擬觀察EP對泡沫細(xì)胞自噬和凋亡的影響,并初步探討可能的機(jī)制。方法：1.THP-1源性泡沫細(xì)胞模型的構(gòu)建：用不同濃度ox-LDL(0mg/L～150mg/L)誘導(dǎo)構(gòu)建THP-1源性泡沫細(xì)胞模型。之后用油紅O染色鑒定造模情況,用MTT法測細(xì)胞生存率。2. ox-LDL刺激對泡沫細(xì)胞自噬和凋亡的影響：THP-1細(xì)胞經(jīng)PMA誘導(dǎo)貼壁后,給予不同濃度的ox-LDL(0mg/L～150mg/L)孵育24小時(shí),用流式細(xì)胞術(shù)檢測細(xì)胞自噬和凋亡水平,以篩選最佳模型構(gòu)建條件。3.EP對THP-1源性泡沫細(xì)胞自噬及凋亡的影響：用上述研究篩選的最適ox-LDL濃度刺激泡沫細(xì)胞生成,應(yīng)用不同濃度EP(5mM-20mM)進(jìn)行干預(yù),觀察其對泡沫細(xì)胞自噬和細(xì)胞凋亡的影響。分組如下：①對照組：不加任何干預(yù)因素：②泡沫細(xì)胞組：加入100mg/L的ox-LDL;③低濃度EP組：加入100mg/L的ox-LDL和5mM的EP；④中濃度EP組：加入100mg/L的ox-LDL和10mM的EP；⑤高濃度EP組：加入100mg/L的ox-LDL和20mmM的EP。共同孵育24小時(shí)后,用western blot、流式細(xì)胞術(shù)、免疫熒光、PCR技術(shù)觀察細(xì)胞自噬水平、細(xì)胞凋亡率。評估不同濃度EP對THP-1源性泡沫細(xì)胞脂質(zhì)蓄積、細(xì)胞自噬和細(xì)胞凋亡的影響。用免疫熒光、PCR技術(shù)評估各組HMGB1水平表達(dá)的差異。4.EP對泡沫細(xì)胞自噬和凋亡影響可能的分子機(jī)制：用免疫熒光、PCR技術(shù)評估ox-LDL組與ox-LDL+20mMEP組HMGB1、Beclin1、Rage等基因水平表達(dá)的差異,探討EP作用于泡沫細(xì)胞自噬和凋亡水平可能的機(jī)制。結(jié)果：1.ox-LDL為25mg/L時(shí),細(xì)胞生存率即下降至87.18%±5.34%(與對照組比較P0.05)。隨著ox-LDL濃度升高,細(xì)胞活力逐漸下降,油紅O染色可見泡沫細(xì)胞內(nèi)大量脂滴。2.THP-1源性泡沫細(xì)胞自噬率及凋亡率水平隨著ox-LDL濃度增高增加,0mgL組,25mg/L組,50mg/L組,100mg/L組,150mg/L組的自噬率分別為0.05%±0.01%,1.28%±0.32%,2.09%±0.17%,5.01%±0.33%,5.41%±0.27%,凋亡率分別為10.01%±0.19%,20.36%±3.42%,29.83±6.27%,49.29%±10.66%,62.70%±1.95%。但100mg/L組與150mg/L組間差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。3.流式細(xì)胞術(shù)、]eal-time PCR. Western blot等檢測手段證實(shí),隨著EP濃度增高,自噬率、自噬相關(guān)基因的表達(dá)水平逐漸下降。泡沫細(xì)胞組凋亡率較對照組明顯增加(48.77±7.22 VS 5.87±1.03)(P0.05)。不同濃度EP組(5mM,10mM,20mM)細(xì)胞凋亡率分別為26.12%±3.99%,12.92%±1.32%,9.46%±0.53%,均較泡沫細(xì)胞組明顯下降(P0.05),凋亡標(biāo)志性蛋白caspase-3表達(dá)隨著EP濃度增加而減少。即隨著EP濃度升高,凋亡率下降明顯。4.免疫熒光染色、PCR顯示ox-LDL+20mMEP組HMGB1、Beclin1、Rage和TNF-a基因mRNA表達(dá)水平較ox-LDL組顯著下降。結(jié)論：1.以PMA誘導(dǎo)THP-1細(xì)胞24小時(shí)可以促使該細(xì)胞貼壁分化為巨噬細(xì)胞；之后再以ox-LDL誘導(dǎo)泡沫細(xì)胞形成。方法簡單可重復(fù)性好。2.在ox-LDL誘導(dǎo)THP-1源性泡沫細(xì)胞中自噬和凋亡水平較對照組均增加。3.不同濃度EP可以抑制泡沫細(xì)胞中的自噬和凋亡,呈濃度依賴性。4.EP可能是通過抑制HMGB1減少了HMGB1-Beclin1互動(dòng)及抑制HMGB1-RAGE通路來減少泡沫細(xì)胞中的自噬；可能是通過HMGB1-TLR4-TNF-α軸通路來抑制凋亡現(xiàn)象。
[Abstract]:Objective: To investigate the effect of macrophages on oxidized low density lipoprotein (ox-LDL) and to convert them into foam cells. The study found that a large number of deaths in the foam cells could result in a significant accumulation of lipid, the progression of the plaque, or even the rupture. The mode of cell death includes autophagic death, apoptosis and necrosis. Ethyl Pyruvate (EP) is a pyruvic acid derivative, and a number of studies show that the drug can play a protective role in organs by inhibiting autophagy and apoptosis of cells and reducing cell death. This study is to observe the effect of EP on autophagy and apoptosis of foam cells and to explore possible mechanisms. Methods:1. Construction of THP-1-derived foam cell model: The model of THP-1-derived foam cells was constructed by different concentration of ox-LDL (0 mg/ L ~ 150 mg/ L). The cell survival rate was measured by MTT method. The effect of ox-LDL on the autophagy and apoptosis of the foam cells was: after the THP-1 cells were induced by PMA, different concentrations of ox-LDL (0 mg/ L-150 mg/ L) were given for 24 hours, and the autophagy and the level of apoptosis were detected by flow cytometry to screen the best model construction conditions. The optimal ox-LDL concentration was used to stimulate the generation of the foam cells and to use different concentrations of EP (5 mM-20 mM) to interfere with the autophagy and apoptosis of the foam cells. The group was as follows: control group: non-foam cell group: add 100 mg/ L of ox-LDL; low-concentration EP group: add 100 mg/ L of ox-LDL and 5 mM of EP; concentration of EP group: add 100 mg/ L of ox-LDL and 10 mM of EP; high-concentration EP group: add 100 mg/ L of ox-LDL and 20 mmM of EP. After 24 hours of incubation, the autophagy level and the cell apoptosis rate were observed by western blot, flow cytometry, immunofluorescence and PCR. The effects of different concentrations of EP on lipid accumulation, autophagy and apoptosis in THP-1-derived foam cells were evaluated. The difference of the expression of HMGB1 in each group was assessed by means of immunofluorescence and PCR.4. The possible molecular mechanism of the effect of EP on autophagy and apoptosis of the foam cells: the difference of the level of HMGB1, Beclin 1, and Rage of the ox-LDL group and the ox-LDL + 20 mMEP group was assessed by immunofluorescence and PCR. The possible mechanisms of EP on autophagy and apoptosis were discussed. Results: The survival rate of the cells decreased to 87.18% and 5.34% when the concentration of 1. oxen-LDL was 25 mg/ L (P0.05). As the concentration of ox-LDL increased, the cell viability gradually decreased, and the oil red O staining showed a large number of lipid droplets in the foam cells.2. The autophagocytosis and the apoptosis rate of the THP-1-derived foam cells increased with the increase of the ox-LDL concentration, the self-phagocytosis of the group of 0 mg/ L,25 mg/ L,50 mg/ L,100 mg/ L and 150 mg/ L was 0.05% and 0.01%, respectively. 1.28%, 0.32%, 2.09%, 0.17%, 5.01%, 0.33%, 5.41%, 0.27%, and the apoptosis rate was 10.01%, 0.19%, 20.36%, 3.42%, 29.83% 6.27%, 49.29%, 10.66%, 62.70% and 1.95%, respectively. However, there was no significant difference between 100 mg/ L and 150 mg/ L (P0.05). Flow cytometry,] eal-time PCR. Western blot and other methods of detection confirmed that with the increase of EP, the self-phagocytosis and autophagy-related genes decreased gradually. The apoptosis rate of the foam cells increased significantly (48.77% 7.22 VS 5.87-1.03) (P0.05). The apoptosis rates of different concentration EP groups (5 mM,10 mM,20 mM) were 26.12%, 3.99%, 12.92%, 1.32%, 9.46% and 0.53%, respectively. In other words, with the increase of EP, the rate of apoptosis was decreased. The expression of HMGB1, Bechin1, Rage and TNF-a gene in the ox-LDL + 20 mMEP group was significantly lower than that of the ox-LDL group. Conclusion:1. The induction of THP-1 cells by PMA for 24 hours can cause the cell to adhere to the macrophages and then induce the formation of the foam cells by ox-LDL. The method is simple and repeatable. The level of autophagy and apoptosis in the THP-1-derived foam cells induced by ox-LDL increased. Different concentrations of EP can inhibit autophagy and apoptosis in the foam cells, in a concentration-dependent manner.4. EP may be to reduce autophagy in the foam cells by inhibiting the HMGB1-Beclo1 interaction and to inhibit the HMGB1-RAGE pathway; it may be the inhibition of apoptosis by the HMGB1-TLR4-TNF-necrosis axis pathway.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R543.5

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