肝源性Hepcidin在脓毒症发生发展中的作用及机制研究

发布时间：2018-05-17 17:24

本文选题：肝源性hepcidin + 尾静脉高动力大剂量注射　；参考：《浙江大学》2015年博士论文

【摘要】：第一部分肝源性hepcidin基因沉默小鼠模型的建立研究目的：阳离子抗菌肽hepcidin是一种主要由肝脏合成分泌的小分子多肽。除了具有微弱的杀菌作用外,hepcidin亦是机体唯一的铁调素,主要参与铁稳态的调节。研究表明,脓毒症病人血清和尿液hepcidin的水平较对照组明显升高。因而推测hepcidin可能在脓毒症的感染和免疫炎症调节过程中发挥重要作用。因此,本部分研究拟采用尾静脉高动力大剂量注射hepcidin特异性干扰腺病毒的方法构建肝源性hepcidin基因沉默小鼠模型,为进一步分析肝源性hepcidin在脓毒症发生发展中的作用奠定基础。研究方法：采用尾静脉高动力大剂量注射的方法,给予balb/c雄性小鼠注射针对小鼠hepcidin基因(hepcl)的特异性shRNA重组腺病毒载体(Ad-shHepcl),对肝脏hepcidin进行基因沉默。对照组采用同样的方法给予对照重组腺病毒(Ad-shNeg)。于注射后13天,取小鼠的外周血、肝、脾、肺和肾脏等进行检测。采用定量PCR方法检测外周血白细胞、肝脏、脾脏、肺脏和肾脏组织hepcidin的mRNA表达水平。采用免疫组织化学的方法检测肝脏、脾脏、肺脏和肾脏组织hepcidin的蛋白表达水平。采用普鲁士蓝染色的方法检测脾脏铁含量。同时,采用原子吸收光谱法测定小鼠血清浓度,酸消化法检测脾脏和肝脏组织的非血红素铁含量。结果：与注射对照重组腺病毒(Ad-shNeg)的小鼠相比较,尾静脉高动力大剂量注射hepcidin特异性shRNA重组腺病毒载体(Ad-shHepcl)的小鼠13天后,肝脏hepcidin的mRNA水平和蛋白水平明显降低。其他组织脏器如白细胞、脾脏、肺脏和肾脏,hepcidin表达水平无明显变化。注射Ad-shHepcl的小鼠,脾脏巨噬细胞铁含量较对照组明显降低,血清铁含量较对照组明显升高。然而,两组肝脏铁含量无明显统计学差异。结论：尾静脉高动力大剂量注射hepcidin特异性shRNA重组腺病毒载体(Ad-shHepcl),能够有效抑制肝脏hepcidin表达,降低脾脏铁含量,增加血清铁水平,成功构建了肝源性hepcidin基因沉默(hepcidin knockdown)小鼠模型。第二部分肝源性hepcidin对脓毒症发生发展的影响研究目的：Hepcidin是一种主要由肝脏合成分泌的,β-defensin样的阳离子抗菌肽,具有广泛的生物学活性。大量研究表明,炎症等刺激能够诱导肝源性hepcidin的表达上调。前期研究显示,危重病患者血浆IL-6的水平与]hepcidin的含量密切相关。临床研究发现,脓毒症病人血清和尿液hepcidin的水平明显升高。鉴于肝源性Hepcidin knockdown小鼠模型的成功建立,因此本部分研究主要探讨肝源性hepcidin对脓毒症发生发展的影响。研究方法：采用盲肠结扎穿孔术(CLP)对肝源性hepcidin knockdown (Ad-shHepcl)小鼠和对照组(Ad-shNeg)小鼠进行脓毒症模型的复制,观察两组小鼠脓毒症的7天生存率。同时,于CLP手术后24小时收集实验组和对照组小鼠的组织样本,如外周血、肝脏、脾脏、肺脏和肾脏等。采用定量PCR和免疫组化法检测小鼠脓毒症后肝脏hepcidin的表达变化。采用苏木素-伊红(HE)染色的方法检测肝脏和肺脏的组织病理变化。采用血清AST和ALT检测的方法评估肝功能的变化。采用原位末端转移酶标记(TUNEL)染色的方法检测脾脏组织的凋亡损伤情况。采用酶联免疫吸附实验(ELISA)检测外周血炎症因子IL-6和TNF-a的水平变化。采用琼脂平板培养法检测小鼠外周血和器官脏器的细菌感染情况。结果：给予CLP脓毒症手术后,肝源性hepcidin knockdown小鼠7天生存率较对照组明显降低。CLP手术24小时后,与对照组相比较,肝源性hepcidin knockdown小鼠肝脏hepcidin的mRNA和蛋白表达水平均明显降低。同时,肝源性hepcidin knockdown小鼠脓毒症后肝脏损伤明显加重,血清ALT和AST水平明显升高。肺组织损伤严重,肺损伤评分明显增高。脾脏凋亡细胞的数量明显增多。肝脏组织的NADPH氧化酶活性亦明显增强,氧化应激损伤加重。此外,脓毒症打击后,肝源性hepcidin knockdown小鼠血浆炎症因子水平较对照组明显降低,外周血及肝脏和肺脏的细菌负荷较对照组明显增多,而两组小鼠脾脏的细菌数量无明显差异。结论：肝源性hepcidin knockdown小鼠脓毒症的死亡率明显增加。给予脓毒症打击后,肝源性hepcidin knockdown小鼠脏器损伤、氧化应激和细菌感染程度均明显加重,免疫反应能力亦明显减弱。肝源性hepcidin在脓毒症感染的免疫防御和预后中可能发挥重要作用。第三部分肝源性hepcidin影响脓毒症发生发展的机制研究研究目的：肝源性hepcidin参与机体铁稳态的调节。感染和炎症等刺激能够诱导hepcidin的表达上调。hepcidin在感染和炎症免疫中可能发挥着至关重要的作用。鉴于肝源性hepcidin knockdown小鼠脓毒症的7天生存率明显降低,因此,本部分研究主要探讨肝源性hepcidin影响脓毒症发生发展的作用机制。研究方法：采用盲肠结扎穿孔手术对肝源性hepcidin knockdown小鼠及对照组小鼠进行脓毒症模型的复制,观察术后24小时小鼠铁代谢变化。采用原子吸收光谱法检测血清铁水平。采用普鲁士蓝染色和酸消化法检测脾脏组织铁含量。同时,给予小鼠巨噬细胞RAW264.7细胞系以不同浓度的铁螯合剂去铁胺(deferoxamine, DFO)处理,通过吞噬荧光颗粒的方法采用荧光显微镜和流式细胞术观察巨噬细胞的吞噬能力,通过脂多糖(LPS)的刺激采用荧光定量PCR的方法检测巨噬细胞的炎症反应能力。最后,对肝源性hepcidin knockdown小鼠给予低铁饮食联合腹腔注射去铁胺的方法处理,13天后检测肝源性hepcidin knockdown小鼠血清铁含量的水平。并且给予低铁处理组小鼠和未给予低铁处理组小鼠以脓毒症的打击,观察两组脓毒症小鼠的7天生存率。结果：脓毒症手术24小时后,与对照组小鼠相比较,肝源性hepcidin knockdown小鼠血清铁含量明显升高,脾脏巨噬细胞的铁含量明显减少。同时,经铁螯合剂处理后的RAW264.7吞噬细胞,其细胞胞内吞噬荧光颗粒的数量及荧光颗粒的强度较未处理细胞均明显降低,其对脂多糖(LPS)刺激后的炎症反应能力亦明显减弱,巨噬细胞炎症因子IL-6的mRNA水平较未处理细胞明显降低。此外,给予低铁饮食联合腹腔注射去铁胺的方法处理肝源性hepcidin knockdown小鼠后,肝源性hepcidin knockdown小鼠血清高铁负荷状态明显改善。给予脓毒症打击后,低铁处理后的肝源性hepcidin knockdown小鼠脓毒症的7天生存率亦明显改善。结论：肝源性hepcidin knockdown小鼠脓毒症后铁代谢紊乱,表现为血清铁水平明显升高,脾脏巨噬细胞铁含量明显减少。胞内铁含量减少明显抑制巨噬细胞的吞噬功能和炎症反应能力。低铁处理肝源性hepcidin knockdown小鼠后可明显改善小鼠的高铁负荷状态,并降低脓毒症的死亡率。其作用机制与hepcidin调节机体铁代谢的功能密切相关。
[Abstract]:Part one establishment of a mouse model of hepatic hepcidin gene silencing
Objective: cationic antibacterial peptide hepcidin is a small molecular polypeptide, which is mainly synthesized and secreted by the liver. In addition to the weak bactericidal effect, hepcidin is the only ferrite in the body, which is mainly involved in the regulation of iron homeostasis. The study shows that the level of serum and urine hepcidin in patients with sepsis is significantly higher than that in the control group. Hepcidin may play an important role in the infection of sepsis and the regulation of immune inflammation. Therefore, this part of this study is to construct a mouse model of hepatgenic hepcidin gene silencing by using hepcidin specific interfering adenovirus with high dose of tail vein, so as to further analyze the development of hepatotoxic hepcidin in sepsis. The role of the medium lays the foundation.
Methods: the balb/c male mice were injected with the specific shRNA recombinant adenovirus vector (Ad-shHepcl) for the mouse hepcidin gene (hepcl) by high dynamic and large dose injection of the tail vein. The hepcidin gene was silenced in the liver. The control group was given the control recombinant adenovirus (Ad-shNeg) by the same method. 13 days after the injection, the control group was given the recombinant adenovirus (Ad-shNeg). Detection of peripheral blood, liver, spleen, lung and kidney of mice. Quantitative PCR method was used to detect the mRNA expression level of hepcidin in peripheral blood white blood cells, liver, spleen, lung and kidney tissue. The protein expression level of hepcidin in liver, spleen, lung and kidney tissues was detected by immunohistochemistry. Prussian blue staining was used. Methods the content of iron in spleen was detected. At the same time, the serum concentration of mice was measured by atomic absorption spectrometry, and the content of non heme iron in spleen and liver tissues was detected by acid digestion.
Results: the mRNA level and protein level of liver hepcidin in the liver of hepcidin specific shRNA recombinant adenovirus vector (Ad-shHepcl) in the tail vein were significantly reduced after 13 days in mice with the injection of recombinant adenovirus (Ad-shNeg). Other tissues such as leukocyte, spleen, lung and kidney, hepcidin expression were found. In mice injected with Ad-shHepcl, the iron content in the spleen macrophages was significantly lower than that in the control group, and the iron content in the serum was significantly higher than that in the control group. However, there was no significant difference in the iron content in the two groups.
Conclusion: the high dose injection of hepcidin specific shRNA recombinant adenovirus vector (Ad-shHepcl) can effectively inhibit the expression of hepcidin in the liver, reduce the iron content of the spleen and increase the level of serum iron, and successfully construct a mouse model of hepatgenic hepcidin gene silencing (hepcidin knockdown).
The second part is the effect of hepatic hepcidin on the development of sepsis.
Objective: Hepcidin is a beta -defensin like cationic antibacterial peptide, which is mainly synthesized and secreted by the liver. A large number of studies have shown that inflammation and other stimuli can induce the up regulation of the expression of hepatderived hepcidin. Previous studies have shown that the level of plasma IL-6 in critically ill patients is closely related to the content of]hepcidin. The bed study found that the levels of serum and urine hepcidin in patients with sepsis were significantly higher. In view of the successful establishment of the hepatderived Hepcidin knockdown mouse model, this part of this study mainly explored the effect of hepatgenic hepcidin on the development of sepsis.
Methods: the cecum ligation and perforation (CLP) was used to replicate the sepsis model in the liver derived hepcidin knockdown (Ad-shHepcl) mice and the control group (Ad-shNeg) and to observe the 7 natural survival rate of sepsis in two groups of mice. At the same time, the tissue samples of the test group and the control group, such as the peripheral blood and the liver, were collected at 24 hours after the CLP operation. Spleen, lung and kidney, and so on. Quantitative PCR and immunohistochemical method were used to detect the changes of liver hepcidin expression in mice with sepsis. The histopathological changes of liver and lung were detected by hematoxylin eosin (HE) staining. The changes of liver function were evaluated by serum AST and ALT. In situ terminal transferase labeling (TUNEL) was used. The staining method was used to detect the apoptosis of spleen tissue. The changes of IL-6 and TNF-a were detected by enzyme linked immunosorbent assay (ELISA). The bacterial infection of peripheral blood and organ organs of mice was detected by agar plate culture.
Results: after the operation of CLP sepsis, the 7 day survival rate of liver derived hepcidin knockdown mice was significantly lower than that of the control group for 24 hours. Compared with the control group, the level of mRNA and protein expression of liver hepcidin in the liver derived hepcidin knockdown mice were significantly decreased. Meanwhile, the liver derived hepcidin knockdown mice were infected with sepsis. The levels of ALT and AST in the serum were significantly increased. The levels of the lung tissue were seriously damaged, the scores of lung injury were significantly increased. The number of apoptotic cells in the spleen increased significantly. The activity of NADPH oxidase in the liver tissues was obviously enhanced and the oxidative stress was aggravated. In addition, after the sepsis was hit, the plasma inflammatory factors of the liver derived hepcidin knockdown mice were in the plasma. The level of bacteria in peripheral blood, liver and lungs was significantly lower than that in the control group, while there was no significant difference in the number of bacteria in the spleen between the two groups.
Conclusion: the death rate of sepsis in the liver derived hepcidin knockdown mice was significantly increased. After the attack of sepsis, the organ damage of the hepcidin knockdown mice, the oxidative stress and the degree of bacterial infection were obviously aggravated, and the immune response ability was obviously weakened. The liver derived hepcidin may be in the immune defense and prognosis of sepsis. Play an important role.
The third part is about the mechanism of hepatocyte derived hepcidin affecting the development of sepsis.
Objective: hepatogenic hepcidin is involved in the regulation of iron homeostasis in the body. Infection and inflammation can induce hepcidin expression up regulation of.Hepcidin in infection and inflammatory immunity. In view of the significant decrease in the 7 natural survival rate of sepsis in hepatderived hepcidin knockdown mice, this part of this study To explore the mechanism of hepatocyte derived hepcidin on the development of sepsis.
Methods: using the cecum ligation and perforation operation to replicate the liver derived hepcidin knockdown mice and the control mice, the changes of iron metabolism in mice were observed 24 hours after the operation. The serum iron levels were detected by atomic absorption spectrometry. The iron content of the spleen was detected by Prussian blue staining and acid digestion. The RAW264.7 cell lines of mouse macrophages were treated with deferoxamine (DFO) with different concentrations of iron chelating mixture. The phagocytosis of macrophages was observed by fluorescence microscopy and flow cytometry by phagocytosis of fluorescent particles. The inflammatory reaction of macrophages was detected by the method of fluorescence quantitative PCR of the lipopolysaccharide (LPS). Finally, the liver derived hepcidin knockdown mice were given a low iron diet combined with intraperitoneal injection of deferamine, and 13 days later, the level of serum iron content in the liver derived hepcidin knockdown mice was detected. And the mice in the low iron treatment group and the group that were not given the low iron treatment group were treated with the attack of sepsis, and the two groups of sepsis were observed. The 7 natural survival rate of the rat.
Results: after 24 hours of sepsis, compared with the control group, the serum iron content of the liver derived hepcidin knockdown mice was significantly increased and the iron content of the spleen macrophages decreased significantly. At the same time, the number of intracellular phagocytic fluorescent particles and the intensity of the fluorescent particles were less than that of the RAW264.7 phagocytes treated by the iron chelating agent. The inflammatory response to lipopolysaccharide (LPS) was significantly reduced, and the mRNA level of macrophage inflammatory factor IL-6 was significantly lower than that of the untreated cells. In addition, the liver derived hepcidin knockdown was small after the low iron diet combined with intraperitoneal injection of DIFERRIC amine in the liver derived hepcidin knockdown mice. The state of high iron load in the rat serum was significantly improved. After the treatment of sepsis, the 7 day survival rate of sepsis in the liver derived hepcidin knockdown mice after low iron treatment was also significantly improved.
Conclusion: the iron metabolism disorder in the liver derived hepcidin knockdown mice showed that the serum iron level was significantly elevated, the iron content of the spleen macrophage decreased obviously. The decrease of intracellular iron content obviously inhibited the phagocytosis and inflammatory response of macrophages. The mice with liver derived hepcidin knockdown could obviously improve the mice after treatment. The high iron load state and reduce the mortality rate of sepsis are closely related to the function of hepcidin regulating the body's iron metabolism.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R459.7

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