中性粒细胞在非酒精性脂肪性肝病中的作用研究

发布时间：2018-04-20 10:50

本文选题：非酒精性脂肪性肝病 + 中性粒细胞　；参考：《安徽医科大学》2017年硕士论文

【摘要】：非酒精性脂肪性肝病(Non-alcoholic Fatty Liver Disease,NAFLD)是一类主要表现为与酒精摄入无关的肝脏脂肪沉积、肝脏脂肪性变的临床病理综合征。据统计,全世界约有10亿人患有NAFLD,在我国有近3亿人患有NAFLD,NAFLD除了可直接导致肝硬化、肝癌外,还参与动脉粥样硬化和II型糖尿病的发病,严重威胁着人类的健康。NAFLD会导致肝细胞受损,损伤和坏死的肝细胞释放损伤相关的模式分子(damage-associated molecular patterns,DAMP),DAMP被免疫系统识别,从而引发炎症反应。本研究通过检测NAFLD患者和健康人的血清中的IL-1β、IL-6和TNF-α的水平,发现NAFLD患者血清中三种细胞因子的水平显著高于健康人,取NAFLD患者外周血上血液分析仪检测,发现NAFLD患者外周血的中性粒细胞的比例显著高于健康人(p0.05)。随后我们构建NAFLD小鼠模型,取外周血上血液分析仪检测,发现NAFLD模型小鼠的外周血中的中性粒细胞的比例显著高于正常小鼠;检测NAFLD模型小鼠的肝脏中的中性粒细胞胞外菌阱(neutrophil extracellular traps,NETs)的水平,发现NAFLD模型小鼠的肝脏中NETs的水平显著升高,检测NAFLD模型小鼠的肝细胞的坏死,发现NAFLD模型小鼠的肝细胞坏死水平高于正常小鼠,分别加入MPO-DNA和Cit-H3的抑制剂后NAFLD模型小鼠的肝细胞坏死水平下降。我们将NAFLD模型小鼠的中性粒细胞与NAFLD模型小鼠的肝脏中的Kuffer细胞共孵育,检测培养基上清中的IL-1β、IL-6和TNF-α的水平,发现3种细胞因子的水平显著升高。为研究NAFLD中损伤的肝细胞通过何种信号通路激活中性粒细胞,导致NETs水平升高,我们将500μg anti-Ly6G抗体注射到NAFLD模型小鼠体内来删除其中性粒细胞,24h后分别将TLR4ko或TLR9ko的中性粒细胞过继到NAFLD模型小鼠体内,24h后检测肝组织中NETs、IL-1β、IL-6和TNF-α的水平,发现过继TLR4ko、TLR9ko的中性粒细胞后的NAFLD模型小鼠的肝脏中NETs和TNF-α的水平均显著下降,肝细胞的凋亡水平下降。本次研究表明,NAFLD模型小鼠中,肝细胞受损后释放DAMP,通过TLR4和TLR9信号通路激活了中性粒细胞,肝组织中NETs水平升高,导致更多的肝细胞坏死,中性粒细胞还能激活Kuffer细胞,促进其IL-1β、IL-6和TNF-α的分泌,从而进一步加重了肝脏的炎性损伤。
[Abstract]:Non-alcoholic Fatty Liver disorder (NAF LDD) is a kind of clinicopathological syndrome characterized by hepatic fatty deposition and hepatic steatosis, which is not related to alcohol intake. According to statistics, there are about 1 billion people in the world suffering from NAF LDD. In our country, nearly 300 million people suffer from NAFLDL NAFLD, which can directly lead to cirrhosis, liver cancer, atherosclerosis and type 2 diabetes. NAFLD is a serious threat to human health. NAFLD can cause hepatocyte damage. The model molecule associated with damage-associated molecular patternsdamp damage-associated molecular pattern damage is recognized by the immune system. In this study, we detected the levels of IL-1 尾 -IL-6 and TNF- 伪 in the serum of NAFLD patients and healthy people, and found that the serum levels of three cytokines in NAFLD patients were significantly higher than those in healthy people. It was found that the percentage of neutrophils in peripheral blood of NAFLD patients was significantly higher than that of healthy controls (P 0.05). Then we constructed the NAFLD mouse model and detected the percentage of neutrophils in the peripheral blood of NAFLD model mice by blood analyzer, and found that the percentage of neutrophils in the peripheral blood of NAFLD model mice was significantly higher than that of normal mice. The level of neutrophil extracellular trapsonic nets in the liver of NAFLD model mice was detected. The level of NETs in the liver of NAFLD model mice was significantly increased, and the necrosis of hepatocytes in NAFLD model mice was detected. It was found that the level of hepatocyte necrosis in NAFLD model mice was higher than that in normal mice, and the level of hepatocyte necrosis in NAFLD model mice decreased after the addition of MPO-DNA and Cit-H3 inhibitors respectively. The neutrophils of NAFLD model mice were incubated with Kuffer cells in liver of NAFLD model mice. The levels of IL-1 尾 -IL-6 and TNF- 伪 in the supernatant of culture medium were detected, and the levels of three cytokines were significantly increased. In order to study the signal pathway that activates neutrophils in the injured hepatocytes of NAFLD, the level of NETs is increased. We injected 500 渭 g anti-Ly6G antibody into NAFLD model mice to remove their neutrophil or TLR9ko neutrophils after 24 hours. After 24 hours, we detected the levels of NETsN-1 尾尾 IL-6 and TNF- 伪 in liver tissue of NAFLD model mice. It was found that the levels of NETs and TNF- 伪 in the liver of NAFLD model mice after TLR4koo TLR9ko neutrophils decreased significantly, while the apoptosis level of hepatocytes decreased. This study showed that in the NAFLD model mice, the liver cells released DAMPand activated the neutrophils through the TLR4 and TLR9 signaling pathway. The level of NETs in the liver tissue increased, leading to more hepatocyte necrosis, and the neutrophil activated the Kuffer cells. Promote the secretion of IL-1 尾 -IL-6 and TNF- 伪, and further aggravate the inflammatory injury of liver.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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