丝氨酸棕榈酰辅酶A转移酶与油酸诱导的肝星状细胞凋亡及自噬的关系

发布时间：2018-07-03 09:26

本文选题：油酸 + 神经酰胺　；参考：《南华大学》2016年硕士论文

【摘要】：目的:探讨神经酰胺从头合成途径及其关键酶丝氨酸棕榈酰辅酶A转移酶(serine palmitoyltransferase,SPT)与油酸诱导的肝星状细胞凋亡及自噬的关系。方法:体外培养HSC-T6细胞,应用不同浓度的油酸(0umol/L、25umol/L、50umol/L、100umol/L、150umol/L)培养HSC-T6细胞24h,采取MTT法检测HSC-T6细胞的增殖活力,选取能抑制细胞增殖又不致细胞死亡的三个浓度作为诱导肝星状细胞的最佳油酸浓度(50umol/L、100umol/L、150umol/L),进入下一步实验。实验分为四组:空白对照组和三个不同浓度的油酸诱导组。以Real-time PCR检测神经酰胺从头合成途径关键酶SPT mRNA的表达,MDC染色法检测肝星状细胞自噬溶酶体的变化,流式细胞仪检测HSC-T6细胞凋亡,Western blot检测调亡相关蛋白Bcl-2、Bax和自噬标志蛋白LC3B、Beclin-1、Atg5的表达。结果:1.油酸诱导组HSC-T6细胞的抑制率明显高于对照组(P0.01),且随着油酸浓度越高,抑制率越高。2.油酸诱导组HSC-T6细胞凋亡率明显高于对照组(P0.01),且油酸浓度越高,细胞凋亡率逐渐增高。3.油酸诱导组HSC-T6细胞自噬泡明显多于对照组,且随着油酸浓度的增多,HSC-T6细胞自噬泡的形成逐渐增多。4.不同浓度油酸诱导的HSC-T6细胞中SPT mRNA表达量与凋亡率之间有明显的正相关性(r=0.963,P0.01)。5.不同浓度油酸诱导的HSC-T6细胞中SPT mRNA表达量与促凋亡蛋白Bax表达水平之间有显著的正相关性(r=0.958,P0.01),与抗凋亡蛋白Bcl-2表达水平之间有显著的负相关性(r=-0.971,P0.01)。6.不同浓度油酸诱导的HSC-T6细胞中SPT mRNA表达量与自噬标志蛋白LC3B表达水平之间有显著的正相关性(r=0.993,P0.01),与自噬标志蛋白Beclin-1表达水平之间有显著的正相关性(r=0.953,P0.01),与自噬标志蛋白Atg5表达水平之间有显著的正相关性(r=0.986,P0.01)结论:1.油酸能抑制HSC-T6细胞的增殖,促进其自噬与凋亡,且具有浓度依赖性。2.神经酰胺从头合成途径关键酶丝氨酸棕榈酰辅酶A转移酶(SPT)与油酸诱导的HSC-T6细胞自噬与凋亡之间呈正相关。
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.2

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