【作者】 张文；

【导师】 傅念；

【作者基本信息】 南华大学 ， 消化内科学（专业学位）， 2016， 硕士

【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(P<0.01),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(P<0.01),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,P<0.01)。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,P<0.01),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,P<0.01)。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,P<0.01),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,P<0.01),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,P<0.01)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。更多还原

【Abstract】 Objective:To investigate The relationship between the de novo ceramide synthesis pathway and oleic acid induced hepatic stellate cell apoptosis and autophagy.Methods: Rat hepatic stellate cells, HSC-T6, were administrated with concentration gradient of oleic acid(0 umol/L 、25umol/L、50umol/L 、100 umol/L、150umol/L). After 24 h, cell viability was measured by cell counting MTT,Select three concentration, less effect on cell activity, as the best concentration of oleic acid induced hepatic stellate cells. Experiment is divided into blank control group and three different concentration of oleic acid induced group.Expression of Serine palmitoyltransferase(SPT) mRNA in HSC-T6 cells was quantified by reverse transcriptase polymerase chain react(RT-PCR). The autophagy lysosomal levels of HSC-T6 cells were measured using MDC staining method,flow cytometric detection of HSC apoptosis, real time PCR and Western blot detection of apoptosis protein Bcl-2, Bax; Western blot detection of apoptosis proteins Bcl-2, Bax and autophagy protein LC3 B, Beclin-1, and autophagy gene Atg5.Results:1.The inhibition rate of HSC-T6 cells in oleic acid induced group was significantly higher than that in control group(P<0.01), and the higher the concentration of oleic acid, the higher the inhibition rate.2. The apoptosis rate of HSC-T6 cells induced by oleic acid group increased significantly higher than that in control group(P<0.01),with increased oleic acid concentration, the apoptosis rate increased gradually.3. The HSC-T6 cells induced by oleic acid group autolysosome significantly more than the control group, and with the increase of the concentration of oleic acid, the formation of HSC-T6 cell autophagy lysosome increased gradually.4. There was a significant positive correlation between SPT mRNA expression 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 SPT mRNA expression and the expression of Pro apoptotic protein Bax in HSC-T6 cells induced by oleic acid at different concentrations(r=0.958, P<0.01), there was a significant negative correlation between SPT expression and anti apoptotic protein Bcl-2 expression in HSC-T6 cells induced by different concentrations of oleic acid(r=-0.971, P<0.01).6.There was a significant positive correlation between SPT mRNA expression and the expression of autophagy mark protein lc3 b in HSC-T6 cells induced by oleic acid at different concentrations(r=0.993, P<0.01), there was a significant positive correlation between SPT expression and the expression of autophagy mark protein beclin-1 in HSC-T6 cells induced by different concentrations of oleic acid(r=0.986, P<0.01).Conclusions:1.Oleic acid can inhibit the proliferation of HSC-T6 cells, promote the autophagy and apoptosis, and has a concentration dependent.2. The relationship between ceramide de novo synthesis pathway key enzyme serine palm acyl coenzyme A transferase(SPT) and Autophagy and apoptosis of HSC-T6 cells induced by oleic acid was positively correlated.更多还原