本文選題：油酸 + 神經(jīng)酰胺　； 參考：《南華大學(xué)》2016年碩士論文

【摘要】：目的:探討神經(jīng)酰胺從頭合成途徑及其關(guān)鍵酶絲氨酸棕櫚酰輔酶A轉(zhuǎn)移酶(serine palmitoyltransferase,SPT)與油酸誘導(dǎo)的肝星狀細(xì)胞凋亡及自噬的關(guān)系。方法:體外培養(yǎng)HSC-T6細(xì)胞,應(yīng)用不同濃度的油酸(0umol/L、25umol/L、50umol/L、100umol/L、150umol/L)培養(yǎng)HSC-T6細(xì)胞24h,采取MTT法檢測HSC-T6細(xì)胞的增殖活力,選取能抑制細(xì)胞增殖又不致細(xì)胞死亡的三個(gè)濃度作為誘導(dǎo)肝星狀細(xì)胞的最佳油酸濃度(50umol/L、100umol/L、150umol/L),進(jìn)入下一步實(shí)驗(yàn)。實(shí)驗(yàn)分為四組:空白對照組和三個(gè)不同濃度的油酸誘導(dǎo)組。以Real-time PCR檢測神經(jīng)酰胺從頭合成途徑關(guān)鍵酶SPT mRNA的表達(dá),MDC染色法檢測肝星狀細(xì)胞自噬溶酶體的變化,流式細(xì)胞儀檢測HSC-T6細(xì)胞凋亡,Western blot檢測調(diào)亡相關(guān)蛋白Bcl-2、Bax和自噬標(biāo)志蛋白LC3B、Beclin-1、Atg5的表達(dá)。結(jié)果:1.油酸誘導(dǎo)組HSC-T6細(xì)胞的抑制率明顯高于對照組(P0.01),且隨著油酸濃度越高,抑制率越高。2.油酸誘導(dǎo)組HSC-T6細(xì)胞凋亡率明顯高于對照組(P0.01),且油酸濃度越高,細(xì)胞凋亡率逐漸增高。3.油酸誘導(dǎo)組HSC-T6細(xì)胞自噬泡明顯多于對照組,且隨著油酸濃度的增多,HSC-T6細(xì)胞自噬泡的形成逐漸增多。4.不同濃度油酸誘導(dǎo)的HSC-T6細(xì)胞中SPT mRNA表達(dá)量與凋亡率之間有明顯的正相關(guān)性(r=0.963,P0.01)。5.不同濃度油酸誘導(dǎo)的HSC-T6細(xì)胞中SPT mRNA表達(dá)量與促凋亡蛋白Bax表達(dá)水平之間有顯著的正相關(guān)性(r=0.958,P0.01),與抗凋亡蛋白Bcl-2表達(dá)水平之間有顯著的負(fù)相關(guān)性(r=-0.971,P0.01)。6.不同濃度油酸誘導(dǎo)的HSC-T6細(xì)胞中SPT mRNA表達(dá)量與自噬標(biāo)志蛋白LC3B表達(dá)水平之間有顯著的正相關(guān)性(r=0.993,P0.01),與自噬標(biāo)志蛋白Beclin-1表達(dá)水平之間有顯著的正相關(guān)性(r=0.953,P0.01),與自噬標(biāo)志蛋白Atg5表達(dá)水平之間有顯著的正相關(guān)性(r=0.986,P0.01)結(jié)論:1.油酸能抑制HSC-T6細(xì)胞的增殖,促進(jìn)其自噬與凋亡,且具有濃度依賴性。2.神經(jīng)酰胺從頭合成途徑關(guān)鍵酶絲氨酸棕櫚酰輔酶A轉(zhuǎn)移酶(SPT)與油酸誘導(dǎo)的HSC-T6細(xì)胞自噬與凋亡之間呈正相關(guān)。
[Abstract]:Aim: to investigate the relationship between ceramide ab initio synthesis pathway, serine palmitoylcoA transferase (serine palmitoyltransferase serine) and oleic acid-induced hepatic stellate cell apoptosis and autophagy. Methods: HSC-T6 cells were cultured in vitro. HSC-T6 cells were cultured with different concentrations of oleic acid (0umolr / L) for 24 h. The proliferation activity of HSC-T6 cells was detected by MTT assay. The optimal concentration of oleic acid to induce hepatic stellate cells (50 umol / L ~ 100 umol/ L ~ (10) 渭 mol / L) without cell death was selected to enter the next experiment. The experiment was divided into four groups: blank control group and three different concentrations of oleic acid induction group. Real-time PCR was used to detect the expression of SPT mRNA in ceramide ab initio synthesis pathway. The changes of autophagy lysosomes in hepatic stellate cells were detected by MDC staining. Flow cytometry was used to detect the expression of apoptosis-related protein Bcl-2 Bax and autophagy marker LC3BnBeclin-1 Atg5 in HSC-T6 cells by Western blot. The result is 1: 1. The inhibition rate of HSC-T6 cells induced by oleic acid was significantly higher than that of control group (P0.01), and the inhibition rate increased with the increase of oleic acid concentration. The apoptosis rate of HSC-T6 cells induced by oleic acid was significantly higher than that of control group (P0.01), and the higher the concentration of oleic acid, the higher the apoptotic rate of HSC-T6 cells. The number of autophagy in HSC-T6 cells induced by oleic acid was significantly higher than that in control group, and the formation of autophagy vesicles in HSC-T6 cells increased gradually with the increase of oleic acid concentration. There was a significant positive correlation between the expression of SPT mRNA and apoptosis rate in HSC-T6 cells induced by oleic acid at different concentrations (r = 0.963, P 0.01) .5. There was a significant positive correlation between the expression of SPT mRNA and the expression level of apoptotic protein Bax in HSC-T6 cells induced by different concentrations of oleic acid (r = 0.958, P0.01), and a significant negative correlation between the expression of SPT mRNA and the expression of anti-apoptotic protein Bcl-2 (r-0.971, P0.01, P0.01). 6, in HSC-T6 cells with different concentrations of oleic acid, the expression of SPT mRNA was positively correlated with the expression of pro-apoptotic protein Bax. There was a significant positive correlation between SPT mRNA expression and LC3B expression level in HSC-T6 cells induced by oleic acid at different concentrations (r = 0.993P 0.01), a significant positive correlation between SPT mRNA expression and the expression level of autophagy marker protein Beclin-1 (r = 0.953 / P0.01), and a significant positive correlation between SPT mRNA expression and autophagy marker protein LC3B expression level (r = 0.953 / P0.01). There was a significant positive correlation between the expression level of white Atg5 (r = 0.986, P 0.01). Conclusion: 1. Oleic acid can inhibit the proliferation of HSC-T6 cells and promote its autophagy and apoptosis in a concentration-dependent manner. There was a positive correlation between serine palmitoyl-coA transferase (SPT) and oleic acid-induced autophagy and apoptosis in HSC-T6 cells induced by oleic acid.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R575.2

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