硒蛋白P在非酒精性脂肪性肝病中的血清学特点和作用机制研究
发布时间：2019-06-14 10:59
【摘要】：背景与目的随着经济发展和生活方式的改变,非酒精性脂肪性肝病(Nonalcoholic fatty liverdisease,NAFLD)正逐渐成为全球发病率最高的慢性肝病之一。目前,我国成人NAFLD患病率约为15-20%。NAFLD成为继病毒性肝炎、酒精性肝病后,肝硬化和肝癌的主要病因,并通过加重糖、脂代谢紊乱,促进代谢性疾病的发生与发展。近年来,肝脏因子与NAFLD等代谢性疾病的关系备受关注。研究表明肝脏因子硒蛋白P(Selenoprotein P,SeP)在NAFLD、2型糖尿病及肥胖患者外周血中升高,且被认为能够促进胰岛素抵抗。然而SeP与NAFLD的关系及其背后的分子机制尚未阐明。因此,本研究拟从国人NAFLD患者中SeP血清学特征和SeP与肝脏脂肪变性的关系入手进行探究,以期为NAFLD的诊断提供新思路,并且进一步阐释NAFLD的发生发展机制。方法为了探究NAFLD患者中SeP血清学特征,我们进行了一项病例对照研究。对象来源于2016年3月至2016年10月期间于浙江大学医学院附属第一医院参加体检的人群。采用调查表询问一般情况及既往病史,并采集其体格检查数据。空腹抽血测定血清学指标,并采用人SeP酶联免疫吸附测定试剂盒测定血清SeP浓度。研究对象肝脏脂变的诊断主要通过腹部B超进行。为进一步探究SeP在NAFLD中的作用机制,体内实验我们选取雄性C57BL/6小鼠,采用高脂饮食喂养,诱导NAFLD动物模型;体外实验选用人肝癌细胞株HepG2细胞,采用400μM棕榈酸刺激24h诱导NAFLD细胞模型。利用荧光实时定量PCR及蛋白免疫印迹等手段检测NAFLD小鼠及细胞模型中SeP的mRNA、蛋白表达变化。体外采用小干扰RNA干扰技术抑制SeP表达,质粒转染技术提高SeP表达,并用SeP纯化重组蛋白培养细胞。利用细胞内甘油三酯含量测定观察SeP表达变化对肝细胞脂肪变性的影响,进一步采用蛋白免疫印迹等分子生物学手段分析SeP对相关信号通路的调控作用。结果病例对照研究共纳入158名研究对象,其中NAFLD患者79例,对照组79例,两组对象之间年龄和性别无显著差异。NAFLD患者血清SeP水平显著高于对照组(13.4±7.0vs.11.1±7.1μg/mL,p0.05)。随血清 SeP 的升高,NAFLD 的检出率呈升高趋势,其中中等SeP组NAFLD检出率显著高于低SeP组(58%vs.38%,p0.05)。而且随着NAFLD严重程度的增加血清SeP水平显著升高;其中,中度NAFLD组血清SeP浓度(13.1±5.7μ/mL)和重度组(15.6±8.1μg/mL)显著高于非NAFLD组SeP浓度(11.1±7.1 μg/mL),值分别小于0.05和0.01。进一步分析提示,血清SeP水平与NAFLD危险因素体重指数(r=0.287,p0.05)、谷丙转氨酶(r=0.275,p0.001)、谷草转氨酶(r=0.199,p0.05)、谷氨酰转肽酶(r=0.231,p0.01)及血清尿酸(r=0.208,p0.01)呈正相关。SeP蛋白在NAFLD小鼠及细胞模型中表达均明显升高,而sepplmRNA在两模型中表达下降。体外小干扰RNA抑制SeP表达能显著降低肝细胞内甘油三酯含量,提高腺苷酸活化蛋白激酶(Adenosine Monophosphate Activated Protein,AMPK)及乙酰辅酶A羧化酶(Acetyl-Co A carboxylase,ACC)磷酸化水平;体外SeP过表达能显著加重肝细胞脂肪变性并降低AMPK及ACC磷酸化水平。采用SeP纯化重组蛋白培养HepG2细胞后,细胞内甘油三酯水平随SeP处理浓度的升高呈上升趋势。结论NAFLD患者中血清SeP水平升高,且SeP与NAFLD患病风险及疾病严重程度有关。血清SeP水平与NAFLD危险因素如体重指数、尿酸等呈正相关。在NAFLD模型中SeP蛋白水平上升,抑制SeP表达能改善肝细胞脂变而高表达SeP则加重肝细胞脂变,推测SeP可能通过AMPK-ACC信号通路对肝细胞脂质代谢进行调控,影响肝细胞脂肪贮积。本次对NAFLD中SeP血清学特征和作用机制的调研可以为未来NAFLD血清学诊断标志物的筛选提供思路,也为NAFLD的治疗提供新靶点。
[Abstract]:BACKGROUND & OBJECTIVE: With the change of economic development and lifestyle, Nonalcoholic fatty liver disease (NAFLD) is becoming one of the most common chronic liver diseases. At present, the prevalence of NAFLD in China is about 15-20%. NAFLD is the main cause of liver cirrhosis and liver cancer following viral hepatitis and alcoholic liver disease. In recent years, the relationship between liver factors and metabolic diseases such as NAFLD is of great concern. The results showed that the liver-factor selenium protein P (SeP) was elevated in the peripheral blood of NAFLD, type 2 diabetes and obese patients and was considered to be able to promote insulin resistance. However, the relationship between SeP and NAFLD and the molecular mechanism behind them have not yet been clarified. Therefore, this study is to explore the relationship between SeP serology and liver steatosis in the patients with NAFLD, with a view to providing a new thought for the diagnosis of NAFLD, and further to explain the development mechanism of NAFLD. Methods To explore the serological characteristics of SeP in NAFLD patients, we conducted a case-control study. The subject was from the first hospital affiliated to Zhejiang University Medical College from March 2016 to October 2016 for the medical examination. The general and past medical history were interrogated using the questionnaire and their physical examination data were collected. Serum SeP concentration was determined by fasting blood drawing and the serum SeP concentration was determined by the human SeP enzyme-linked immunosorbent assay (ELISA). The diagnosis of liver lipid changes of the subject was mainly performed by abdominal B-ultrasound. In order to further explore the mechanism of the action of SeP in NAFLD, we selected male C57BL/6 mice by high-fat diet, and induced NAFLD animal model; in vitro, human liver cancer cell line HepG2 cells were selected, and the NAFLD cell model was induced with 400 & mu; M palmitic acid for 24 h. MRNA and protein expression of SeP in NAFLD mice and cell models were detected by fluorescence real-time quantitative PCR and protein immunoblotting. In vitro, the expression of SeP was inhibited by small interfering RNA interference technique, and the expression of SeP was improved by plasmid transfection, and the recombinant protein was purified by SeP. The effect of the expression of SeP on the fatty degeneration of the hepatocytes was observed by the determination of the content of triglyceride in the cells, and the regulatory effects of the SeP on the related signal pathways were further analyzed by using the molecular biological methods such as protein immunoblotting. Results A total of 158 subjects were included in the case-control study,79 of the patients with NAFLD and 79 in the control group, and there was no significant difference between the two groups. The serum level of SeP in the patients with NAFLD was significantly higher than that in the control group (13.4% 7.0 vs. 11.1, 7.1. mu.g/ mL, p0.05). The positive rate of NAFLD in the middle-SeP group was higher than that of the low-SeP group (58% vs.38%, p0.05). The serum SeP level in the moderate NAFLD group was significantly higher than that in the non-NAFLD group (11.1, 7.1. mu.g/ mL), and the values were less than 0.05 and 0.01, respectively, as the level of NAFLD was increased significantly. The body weight index (r = 0.287, p0.05), glutamic-pyruvic transaminase (r = 0.275, p0.001), aspartate aminotransferase (r = 0.199, p0.01), and serum uric acid (r = 0.208, p0.01) were positively correlated with the risk factors of NAFLD (r = 0.287, p0.05), glutamic-pyruvic transaminase (r = 0.275, p0.001), and aspartate aminotransferase (r = 0.199, p0.01) and serum uric acid (r = 0.208, p0.01). The expression of SeP protein in NAFLD mice and cell model was significantly increased, while the expression of seppl mRNA in both models decreased. The inhibition of SeP expression by small interfering RNA in vitro could significantly lower the content of triglyceride in the hepatocytes, increase the level of the phosphorylation of adenosine-activated protein kinase (AMPK) and acetyl-Co A carboxase (ACC). In vitro SeP overexpression can significantly increase the hepatic steatosis and decrease the level of AMPK and ACC phosphorylation. After the HepG2 cells were cultured with SeP purified recombinant protein, the level of triglyceride in the cells increased with the increase of the treatment concentration of SeP. Conclusion The serum level of SeP in the patients with NAFLD is increased, and the risk of SeP and NAFLD is related to the severity of the disease. Serum SeP levels were positively correlated with the risk factors of NAFLD, such as body weight index and uric acid. In the NAFLD model, the level of SeP protein is increased, and the inhibition of SeP expression can improve the liver cell lipid change and the high expression of the SeP increases the liver cell lipid change, and it is presumed that the SeP can regulate the lipid metabolism of the liver cell through the AMPK-ACC signal path, and influence the fat storage of the liver cell. The investigation of the serological characteristics and the mechanism of the SeP in the NAFLD can provide a new way for the screening of the serological diagnostic markers of the NAFLD, and also provide a new target for the treatment of NAFLD.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575
本文编号：2499341
[Abstract]:BACKGROUND & OBJECTIVE: With the change of economic development and lifestyle, Nonalcoholic fatty liver disease (NAFLD) is becoming one of the most common chronic liver diseases. At present, the prevalence of NAFLD in China is about 15-20%. NAFLD is the main cause of liver cirrhosis and liver cancer following viral hepatitis and alcoholic liver disease. In recent years, the relationship between liver factors and metabolic diseases such as NAFLD is of great concern. The results showed that the liver-factor selenium protein P (SeP) was elevated in the peripheral blood of NAFLD, type 2 diabetes and obese patients and was considered to be able to promote insulin resistance. However, the relationship between SeP and NAFLD and the molecular mechanism behind them have not yet been clarified. Therefore, this study is to explore the relationship between SeP serology and liver steatosis in the patients with NAFLD, with a view to providing a new thought for the diagnosis of NAFLD, and further to explain the development mechanism of NAFLD. Methods To explore the serological characteristics of SeP in NAFLD patients, we conducted a case-control study. The subject was from the first hospital affiliated to Zhejiang University Medical College from March 2016 to October 2016 for the medical examination. The general and past medical history were interrogated using the questionnaire and their physical examination data were collected. Serum SeP concentration was determined by fasting blood drawing and the serum SeP concentration was determined by the human SeP enzyme-linked immunosorbent assay (ELISA). The diagnosis of liver lipid changes of the subject was mainly performed by abdominal B-ultrasound. In order to further explore the mechanism of the action of SeP in NAFLD, we selected male C57BL/6 mice by high-fat diet, and induced NAFLD animal model; in vitro, human liver cancer cell line HepG2 cells were selected, and the NAFLD cell model was induced with 400 & mu; M palmitic acid for 24 h. MRNA and protein expression of SeP in NAFLD mice and cell models were detected by fluorescence real-time quantitative PCR and protein immunoblotting. In vitro, the expression of SeP was inhibited by small interfering RNA interference technique, and the expression of SeP was improved by plasmid transfection, and the recombinant protein was purified by SeP. The effect of the expression of SeP on the fatty degeneration of the hepatocytes was observed by the determination of the content of triglyceride in the cells, and the regulatory effects of the SeP on the related signal pathways were further analyzed by using the molecular biological methods such as protein immunoblotting. Results A total of 158 subjects were included in the case-control study,79 of the patients with NAFLD and 79 in the control group, and there was no significant difference between the two groups. The serum level of SeP in the patients with NAFLD was significantly higher than that in the control group (13.4% 7.0 vs. 11.1, 7.1. mu.g/ mL, p0.05). The positive rate of NAFLD in the middle-SeP group was higher than that of the low-SeP group (58% vs.38%, p0.05). The serum SeP level in the moderate NAFLD group was significantly higher than that in the non-NAFLD group (11.1, 7.1. mu.g/ mL), and the values were less than 0.05 and 0.01, respectively, as the level of NAFLD was increased significantly. The body weight index (r = 0.287, p0.05), glutamic-pyruvic transaminase (r = 0.275, p0.001), aspartate aminotransferase (r = 0.199, p0.01), and serum uric acid (r = 0.208, p0.01) were positively correlated with the risk factors of NAFLD (r = 0.287, p0.05), glutamic-pyruvic transaminase (r = 0.275, p0.001), and aspartate aminotransferase (r = 0.199, p0.01) and serum uric acid (r = 0.208, p0.01). The expression of SeP protein in NAFLD mice and cell model was significantly increased, while the expression of seppl mRNA in both models decreased. The inhibition of SeP expression by small interfering RNA in vitro could significantly lower the content of triglyceride in the hepatocytes, increase the level of the phosphorylation of adenosine-activated protein kinase (AMPK) and acetyl-Co A carboxase (ACC). In vitro SeP overexpression can significantly increase the hepatic steatosis and decrease the level of AMPK and ACC phosphorylation. After the HepG2 cells were cultured with SeP purified recombinant protein, the level of triglyceride in the cells increased with the increase of the treatment concentration of SeP. Conclusion The serum level of SeP in the patients with NAFLD is increased, and the risk of SeP and NAFLD is related to the severity of the disease. Serum SeP levels were positively correlated with the risk factors of NAFLD, such as body weight index and uric acid. In the NAFLD model, the level of SeP protein is increased, and the inhibition of SeP expression can improve the liver cell lipid change and the high expression of the SeP increases the liver cell lipid change, and it is presumed that the SeP can regulate the lipid metabolism of the liver cell through the AMPK-ACC signal path, and influence the fat storage of the liver cell. The investigation of the serological characteristics and the mechanism of the SeP in the NAFLD can provide a new way for the screening of the serological diagnostic markers of the NAFLD, and also provide a new target for the treatment of NAFLD.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575
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相关期刊论文 前4条
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2 Ren-Nan Feng;Shan-Shan Du;Cheng Wang;Yan-Chuan Li;Li-Yan Liu;Fu-Chuan Guo;Chang-Hao Sun;;Lean-non-alcoholic fatty liver disease increases risk for metabolic disorders in a normal weight Chinese population[J];World Journal of Gastroenterology;2014年47期
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