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1、上海交通大学 硕士学位论文 脂肪酸对肝细胞损伤机制研究内质网应激及干预 姓名：丁佳 申请学位级别：硕士 专业：内科学（消化系病） 指导教师：范竹萍 20090430 上海交通大学医学院硕士学位论文上海交通大学医学院硕士学位论文 I 脂肪酸对肝细胞损伤机制研究内质网应激及干预 摘 要 背景： 背景：胰岛素抵抗（IR）和中央性肥胖是 NAFLD 的重要发病机制， 同时由中央性肥胖、2 型糖尿病、胰岛素抵抗、高血压及血脂异常等组 成的代谢综合征（metabolic syndrome，MS）是心血管疾病（CVD）的 高危险因素。以往肝脏组织脂肪沉积常常被视为一种良性疾病，但当过 多的甘油三醋（TG）水
2、解成游离脂肪酸（FFA）分流到非脂肪组织 肝脏，则可能会引发肝细胞功能障碍及肝脏组织胰岛素抵抗，此即脂毒 性在肝脏中的表现。内质网（ER）是蛋白质合成、折叠、运输以及细胞 内钙离子的储存场所。多种因素如糖类和营养物质的缺乏、病毒感染、 脂质代谢异常、钙失衡、自由基及药物等均可引起内质网应激。近年来， 大量细胞实验和动物模型表明：肥胖和高脂饮食能诱发肝细胞内质网应 激，并同时伴有肝脏和脂肪组织的胰岛素抵抗。因此，研究脂毒性和内 质网应激在非酒精性脂肪性肝病发病机制中所起的作用，可对代谢综合 征行更深一步的阐述。牛磺熊去氧胆酸作为化学性分子伴侣，能减轻内 质网应激，改善遗传性或饮食诱导的肥胖小鼠模
3、型胰岛素的敏感性。本 课题将通过建立脂肪变肝细胞模型对脂毒性和内质网应激在 NAFLD 发 病机制中的作用和分子伴侣治疗脂肪变性肝细胞的疗效进行深入探讨。 上海交通大学医学院硕士学位论文上海交通大学医学院硕士学位论文 II 第一部分 通过建立肝细胞脂肪变模型探讨不同种类脂肪酸对 肝细胞的作用 目的：目的：通过建立肝细胞脂肪变模型探讨不同种类脂肪酸对肝细胞的 作用。方法：方法：将细胞分为三组：对照组、混合脂肪酸组（油酸：软脂酸 =2:1）和软脂酸组（油酸：软脂酸=0:3） ，分别给予不含脂肪酸及含各浓 度脂肪酸的 RPMI-1640 培养液， 用 CCK-8 法测定细胞活力。 并以 0.5mM
4、和 1.0mM 的混合脂肪酸与软脂酸分别培养细胞 12 小时、24 小时、48 小 时，油红 O 染色观察脂肪酸处理后细胞形态及胞浆内脂滴变化情况。用 细胞裂解液裂解细胞后， 酶比色法测定细胞内甘油三酯 （triglyceride, TG） 和胆固醇（total cholesterol, TC）含量。结果：结果：两种脂肪酸都可诱导肝细 胞脂肪变，但单纯软脂酸较混合脂肪酸对正常肝细胞的生长影响更大。 且软脂酸 0.5mM 作用肝细胞 24 小时即可产生肝细胞脂肪变性、坏死； 而混合脂肪酸 1.0mM 作用肝细胞 48 小时后，虽然脂肪变性逐渐加重， 但细胞未见明显坏死。油红 O 染色细胞内出现明
5、显的脂质堆积，脂滴数 增多；随造模时间延长，混合脂肪酸各组细胞内甘油三酯含量均高于对 照组（P 0.05） ； 软脂酸组 （油 酸：软脂酸 = 0:3）GRP78和CHOPmRNA的表达量随着作用时间的延长 而增加， 24h到达高峰后开始下降。 相同的脂肪酸浓度前提下， 0.5mM 时 GRP78mRNA表达量在两组脂肪变模型间差异不显著（P=0.870） ；而 CHOPmRNA表达量差异却十分显著（P 0.001） ；1.0mM时软脂酸组 GRP78和CHOPmRNA两者的表达量都显著增加（P 均 0.05） ；对 1.0mM 软脂酸引起的 ERS 时 GRP78、 CHOPmRNA 表达量
6、增加有改善作用（12h：P=0.066 vs P=0.042；24h： P=0.053 vs P=0.017） 。结论：结论：TUDCA 对软脂酸所引起的肝细胞内质网应 激有改善作用。 关键词：关键词： 肝细胞脂肪变性，内质网应激，饱和脂肪酸，牛磺熊去氧 胆酸 上海交通大学医学院硕士学位论文上海交通大学医学院硕士学位论文 IV EFFECT OF FREE FATTY ACIDS ON HEPATOCYTES ENDOPLASMIC RETICULUM STRESS AND INTERVENTION ABSTRACT Backgrounds: Insulin resistance and tr
7、uncal obesity play a critical role in the pathogenesis of NAFLD. Abdominal obesity, type 2 diabetes, insulin resistance, hypertension and dyslipidaemiathe typical components of the metabolic syndrome (MS)are risk factors for cardiovascular disease (CVD). Hepatic steatosis was believed to be a benign
8、 condition, until overacccumulation of lipids in the nonadipose tissue leads to cell dysfunction and hepatic insulin resistance. That is lipotoxicity in hepatocytes. The endoplasmic reticulum (ER) is one of the largest cellular organelles, where proteins are synthesized, posttranslational modificati
9、ons are made and disulfide formation adjusts conformation.Another important function of the ER is to store calcium, which is released in a controlled fashion by various signaling events. Perturbations that lead to elevated protein synthesis, overexpression and/or accumulation of mutant malfolded pro
10、teins, glucose deprivation and altered glycosylation, dysregulation of lipid metabolism, ER calcium depletion, and shifting of redox status to more reduced state are sensed by the ER as a need to adapt to handle stress. Recent evidence suggests that endoplasmic reticulum stress links obesity, insuli
11、n action, and type 2 diabetes. But less study has focused on lipotoxicity and ERS in the pathogenesis of NAFLD and their effects on metabolic regulation. Chemical or pharmaceutical chaperones are a group of low molecular weight 上海交通大学医学院硕士学位论文上海交通大学医学院硕士学位论文 V compounds known to stabilize protein co
12、nformation, improve ER folding capacity, and facilitate the trafficking of mutant proteins. Likewise, endogenous bile acids and derivatives such as ursodeoxycholic acid and its taurine-conjugated derivative (TUDCA) can also modulate ER function and improve insulin action in ob/ob mice. In order to k
13、now whether NAFLD involves lipotoxicity and ER stress, we investigated the change of them in cultured steatotic hepatocytes incubated with different fatty acids and assessed the effect of TUDCA on it. Part Fat-overloading induction in HL-7702 cells Objective: Fat accumulation, cytotoxicity and apopt
14、osis in hepatocytes exposed to different FFAs were investigated. Methods: L02 cells were incubated with FFA mixture containing a low proportion of palmitic acid (oleate/palmitate,2:1 ratio) or a high proportion of palmitic acid (oleate/palmitate,0:3 ratio) to induce fat-overloading. Both FFA mixture
15、 at concentrations of 0.5mM and 1.0mM were added to L02 cells 12h, 24h and 48h after seeding, respectively. The lipid content in cultured cells was observed by Oil Red O staining and determined by colorimetric assay. Results: After treating L02 cells with two kinds of FFA mixture 12 hours, all group
16、s showed significantly increased red fatty droplets compared with control group by Oil red O staining. Compared with oleate/palmitate at 2:1 ratio, lower fat level was found in cells treated with palmitate alone (0:3 ratio) after 24h as it was apparently toxic at such concentrations. Moreover, 1.0mM palmitate (0:3 ratio) was shown to be highly toxic after 48h. Oleate/palmitate at 2:1 ratio FFAs overloaded L02 cells accumulated a higher fat content than palmitate alone. Conclusions
