硒蛋白P在非酒精性脂肪性肝病中的血清學(xué)特點(diǎn)和作用機(jī)制研究
發(fā)布時間：2019-06-14 10:59
【摘要】：背景與目的隨著經(jīng)濟(jì)發(fā)展和生活方式的改變,非酒精性脂肪性肝病(Nonalcoholic fatty liverdisease,NAFLD)正逐漸成為全球發(fā)病率最高的慢性肝病之一。目前,我國成人NAFLD患病率約為15-20%。NAFLD成為繼病毒性肝炎、酒精性肝病后,肝硬化和肝癌的主要病因,并通過加重糖、脂代謝紊亂,促進(jìn)代謝性疾病的發(fā)生與發(fā)展。近年來,肝臟因子與NAFLD等代謝性疾病的關(guān)系備受關(guān)注。研究表明肝臟因子硒蛋白P(Selenoprotein P,SeP)在NAFLD、2型糖尿病及肥胖患者外周血中升高,且被認(rèn)為能夠促進(jìn)胰島素抵抗。然而SeP與NAFLD的關(guān)系及其背后的分子機(jī)制尚未闡明。因此,本研究擬從國人NAFLD患者中SeP血清學(xué)特征和SeP與肝臟脂肪變性的關(guān)系入手進(jìn)行探究,以期為NAFLD的診斷提供新思路,并且進(jìn)一步闡釋NAFLD的發(fā)生發(fā)展機(jī)制。方法為了探究NAFLD患者中SeP血清學(xué)特征,我們進(jìn)行了一項(xiàng)病例對照研究。對象來源于2016年3月至2016年10月期間于浙江大學(xué)醫(yī)學(xué)院附屬第一醫(yī)院參加體檢的人群。采用調(diào)查表詢問一般情況及既往病史,并采集其體格檢查數(shù)據(jù)。空腹抽血測定血清學(xué)指標(biāo),并采用人SeP酶聯(lián)免疫吸附測定試劑盒測定血清SeP濃度。研究對象肝臟脂變的診斷主要通過腹部B超進(jìn)行。為進(jìn)一步探究SeP在NAFLD中的作用機(jī)制,體內(nèi)實(shí)驗(yàn)我們選取雄性C57BL/6小鼠,采用高脂飲食喂養(yǎng),誘導(dǎo)NAFLD動物模型;體外實(shí)驗(yàn)選用人肝癌細(xì)胞株HepG2細(xì)胞,采用400μM棕櫚酸刺激24h誘導(dǎo)NAFLD細(xì)胞模型。利用熒光實(shí)時定量PCR及蛋白免疫印跡等手段檢測NAFLD小鼠及細(xì)胞模型中SeP的mRNA、蛋白表達(dá)變化。體外采用小干擾RNA干擾技術(shù)抑制SeP表達(dá),質(zhì)粒轉(zhuǎn)染技術(shù)提高SeP表達(dá),并用SeP純化重組蛋白培養(yǎng)細(xì)胞。利用細(xì)胞內(nèi)甘油三酯含量測定觀察SeP表達(dá)變化對肝細(xì)胞脂肪變性的影響,進(jìn)一步采用蛋白免疫印跡等分子生物學(xué)手段分析SeP對相關(guān)信號通路的調(diào)控作用。結(jié)果病例對照研究共納入158名研究對象,其中NAFLD患者79例,對照組79例,兩組對象之間年齡和性別無顯著差異。NAFLD患者血清SeP水平顯著高于對照組(13.4±7.0vs.11.1±7.1μg/mL,p0.05)。隨血清 SeP 的升高,NAFLD 的檢出率呈升高趨勢,其中中等SeP組NAFLD檢出率顯著高于低SeP組(58%vs.38%,p0.05)。而且隨著NAFLD嚴(yán)重程度的增加血清SeP水平顯著升高;其中,中度NAFLD組血清SeP濃度(13.1±5.7μ/mL)和重度組(15.6±8.1μg/mL)顯著高于非NAFLD組SeP濃度(11.1±7.1 μg/mL),值分別小于0.05和0.01。進(jìn)一步分析提示,血清SeP水平與NAFLD危險因素體重指數(shù)(r=0.287,p0.05)、谷丙轉(zhuǎn)氨酶(r=0.275,p0.001)、谷草轉(zhuǎn)氨酶(r=0.199,p0.05)、谷氨酰轉(zhuǎn)肽酶(r=0.231,p0.01)及血清尿酸(r=0.208,p0.01)呈正相關(guān)。SeP蛋白在NAFLD小鼠及細(xì)胞模型中表達(dá)均明顯升高,而sepplmRNA在兩模型中表達(dá)下降。體外小干擾RNA抑制SeP表達(dá)能顯著降低肝細(xì)胞內(nèi)甘油三酯含量,提高腺苷酸活化蛋白激酶(Adenosine Monophosphate Activated Protein,AMPK)及乙酰輔酶A羧化酶(Acetyl-Co A carboxylase,ACC)磷酸化水平;體外SeP過表達(dá)能顯著加重肝細(xì)胞脂肪變性并降低AMPK及ACC磷酸化水平。采用SeP純化重組蛋白培養(yǎng)HepG2細(xì)胞后,細(xì)胞內(nèi)甘油三酯水平隨SeP處理濃度的升高呈上升趨勢。結(jié)論NAFLD患者中血清SeP水平升高,且SeP與NAFLD患病風(fēng)險及疾病嚴(yán)重程度有關(guān)。血清SeP水平與NAFLD危險因素如體重指數(shù)、尿酸等呈正相關(guān)。在NAFLD模型中SeP蛋白水平上升,抑制SeP表達(dá)能改善肝細(xì)胞脂變而高表達(dá)SeP則加重肝細(xì)胞脂變,推測SeP可能通過AMPK-ACC信號通路對肝細(xì)胞脂質(zhì)代謝進(jìn)行調(diào)控,影響肝細(xì)胞脂肪貯積。本次對NAFLD中SeP血清學(xué)特征和作用機(jī)制的調(diào)研可以為未來NAFLD血清學(xué)診斷標(biāo)志物的篩選提供思路,也為NAFLD的治療提供新靶點(diǎn)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R575
本文編號：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R575
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
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