本文選題：肝源性hepcidin + 尾靜脈高動力大劑量注射　； 參考：《浙江大學(xué)》2015年博士論文

【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：陽離子抗菌肽hepcidin是一種主要由肝臟合成分泌的小分子多肽。除了具有微弱的殺菌作用外,hepcidin亦是機體唯一的鐵調(diào)素,主要參與鐵穩(wěn)態(tài)的調(diào)節(jié)。研究表明,膿毒癥病人血清和尿液hepcidin的水平較對照組明顯升高。因而推測hepcidin可能在膿毒癥的感染和免疫炎癥調(diào)節(jié)過程中發(fā)揮重要作用。因此,本部分研究擬采用尾靜脈高動力大劑量注射hepcidin特異性干擾腺病毒的方法構(gòu)建肝源性hepcidin基因沉默小鼠模型,為進一步分析肝源性hepcidin在膿毒癥發(fā)生發(fā)展中的作用奠定基礎(chǔ)。 研究方法：采用尾靜脈高動力大劑量注射的方法,給予balb/c雄性小鼠注射針對小鼠hepcidin基因(hepcl)的特異性shRNA重組腺病毒載體(Ad-shHepcl),對肝臟hepcidin進行基因沉默。對照組采用同樣的方法給予對照重組腺病毒(Ad-shNeg)。于注射后13天,取小鼠的外周血、肝、脾、肺和腎臟等進行檢測。采用定量PCR方法檢測外周血白細胞、肝臟、脾臟、肺臟和腎臟組織hepcidin的mRNA表達水平。采用免疫組織化學(xué)的方法檢測肝臟、脾臟、肺臟和腎臟組織hepcidin的蛋白表達水平。采用普魯士藍染色的方法檢測脾臟鐵含量。同時,采用原子吸收光譜法測定小鼠血清濃度,酸消化法檢測脾臟和肝臟組織的非血紅素鐵含量。 結(jié)果：與注射對照重組腺病毒(Ad-shNeg)的小鼠相比較,尾靜脈高動力大劑量注射hepcidin特異性shRNA重組腺病毒載體(Ad-shHepcl)的小鼠13天后,肝臟hepcidin的mRNA水平和蛋白水平明顯降低。其他組織臟器如白細胞、脾臟、肺臟和腎臟,hepcidin表達水平無明顯變化。注射Ad-shHepcl的小鼠,脾臟巨噬細胞鐵含量較對照組明顯降低,血清鐵含量較對照組明顯升高。然而,兩組肝臟鐵含量無明顯統(tǒng)計學(xué)差異。 結(jié)論：尾靜脈高動力大劑量注射hepcidin特異性shRNA重組腺病毒載體(Ad-shHepcl),能夠有效抑制肝臟hepcidin表達,降低脾臟鐵含量,增加血清鐵水平,成功構(gòu)建了肝源性hepcidin基因沉默(hepcidin knockdown)小鼠模型。 第二部分肝源性hepcidin對膿毒癥發(fā)生發(fā)展的影響 研究目的：Hepcidin是一種主要由肝臟合成分泌的,β-defensin樣的陽離子抗菌肽,具有廣泛的生物學(xué)活性。大量研究表明,炎癥等刺激能夠誘導(dǎo)肝源性hepcidin的表達上調(diào)。前期研究顯示,危重病患者血漿IL-6的水平與]hepcidin的含量密切相關(guān)。臨床研究發(fā)現(xiàn),膿毒癥病人血清和尿液hepcidin的水平明顯升高。鑒于肝源性Hepcidin knockdown小鼠模型的成功建立,因此本部分研究主要探討肝源性hepcidin對膿毒癥發(fā)生發(fā)展的影響。 研究方法：采用盲腸結(jié)扎穿孔術(shù)(CLP)對肝源性hepcidin knockdown (Ad-shHepcl)小鼠和對照組(Ad-shNeg)小鼠進行膿毒癥模型的復(fù)制,觀察兩組小鼠膿毒癥的7天生存率。同時,于CLP手術(shù)后24小時收集實驗組和對照組小鼠的組織樣本,如外周血、肝臟、脾臟、肺臟和腎臟等。采用定量PCR和免疫組化法檢測小鼠膿毒癥后肝臟hepcidin的表達變化。采用蘇木素-伊紅(HE)染色的方法檢測肝臟和肺臟的組織病理變化。采用血清AST和ALT檢測的方法評估肝功能的變化。采用原位末端轉(zhuǎn)移酶標記(TUNEL)染色的方法檢測脾臟組織的凋亡損傷情況。采用酶聯(lián)免疫吸附實驗(ELISA)檢測外周血炎癥因子IL-6和TNF-a的水平變化。采用瓊脂平板培養(yǎng)法檢測小鼠外周血和器官臟器的細菌感染情況。 結(jié)果：給予CLP膿毒癥手術(shù)后,肝源性hepcidin knockdown小鼠7天生存率較對照組明顯降低。CLP手術(shù)24小時后,與對照組相比較,肝源性hepcidin knockdown小鼠肝臟hepcidin的mRNA和蛋白表達水平均明顯降低。同時,肝源性hepcidin knockdown小鼠膿毒癥后肝臟損傷明顯加重,血清ALT和AST水平明顯升高。肺組織損傷嚴重,肺損傷評分明顯增高。脾臟凋亡細胞的數(shù)量明顯增多。肝臟組織的NADPH氧化酶活性亦明顯增強,氧化應(yīng)激損傷加重。此外,膿毒癥打擊后,肝源性hepcidin knockdown小鼠血漿炎癥因子水平較對照組明顯降低,外周血及肝臟和肺臟的細菌負荷較對照組明顯增多,而兩組小鼠脾臟的細菌數(shù)量無明顯差異。 結(jié)論：肝源性hepcidin knockdown小鼠膿毒癥的死亡率明顯增加。給予膿毒癥打擊后,肝源性hepcidin knockdown小鼠臟器損傷、氧化應(yīng)激和細菌感染程度均明顯加重,免疫反應(yīng)能力亦明顯減弱。肝源性hepcidin在膿毒癥感染的免疫防御和預(yù)后中可能發(fā)揮重要作用。 第三部分肝源性hepcidin影響膿毒癥發(fā)生發(fā)展的機制研究 研究目的：肝源性hepcidin參與機體鐵穩(wěn)態(tài)的調(diào)節(jié)。感染和炎癥等刺激能夠誘導(dǎo)hepcidin的表達上調(diào)。hepcidin在感染和炎癥免疫中可能發(fā)揮著至關(guān)重要的作用。鑒于肝源性hepcidin knockdown小鼠膿毒癥的7天生存率明顯降低,因此,本部分研究主要探討肝源性hepcidin影響膿毒癥發(fā)生發(fā)展的作用機制。 研究方法：采用盲腸結(jié)扎穿孔手術(shù)對肝源性hepcidin knockdown小鼠及對照組小鼠進行膿毒癥模型的復(fù)制,觀察術(shù)后24小時小鼠鐵代謝變化。采用原子吸收光譜法檢測血清鐵水平。采用普魯士藍染色和酸消化法檢測脾臟組織鐵含量。同時,給予小鼠巨噬細胞RAW264.7細胞系以不同濃度的鐵螯合劑去鐵胺(deferoxamine, DFO)處理,通過吞噬熒光顆粒的方法采用熒光顯微鏡和流式細胞術(shù)觀察巨噬細胞的吞噬能力,通過脂多糖(LPS)的刺激采用熒光定量PCR的方法檢測巨噬細胞的炎癥反應(yīng)能力。最后,對肝源性hepcidin knockdown小鼠給予低鐵飲食聯(lián)合腹腔注射去鐵胺的方法處理,13天后檢測肝源性hepcidin knockdown小鼠血清鐵含量的水平。并且給予低鐵處理組小鼠和未給予低鐵處理組小鼠以膿毒癥的打擊,觀察兩組膿毒癥小鼠的7天生存率。 結(jié)果：膿毒癥手術(shù)24小時后,與對照組小鼠相比較,肝源性hepcidin knockdown小鼠血清鐵含量明顯升高,脾臟巨噬細胞的鐵含量明顯減少。同時,經(jīng)鐵螯合劑處理后的RAW264.7吞噬細胞,其細胞胞內(nèi)吞噬熒光顆粒的數(shù)量及熒光顆粒的強度較未處理細胞均明顯降低,其對脂多糖(LPS)刺激后的炎癥反應(yīng)能力亦明顯減弱,巨噬細胞炎癥因子IL-6的mRNA水平較未處理細胞明顯降低。此外,給予低鐵飲食聯(lián)合腹腔注射去鐵胺的方法處理肝源性hepcidin knockdown小鼠后,肝源性hepcidin knockdown小鼠血清高鐵負荷狀態(tài)明顯改善。給予膿毒癥打擊后,低鐵處理后的肝源性hepcidin knockdown小鼠膿毒癥的7天生存率亦明顯改善。 結(jié)論：肝源性hepcidin knockdown小鼠膿毒癥后鐵代謝紊亂,表現(xiàn)為血清鐵水平明顯升高,脾臟巨噬細胞鐵含量明顯減少。胞內(nèi)鐵含量減少明顯抑制巨噬細胞的吞噬功能和炎癥反應(yīng)能力。低鐵處理肝源性hepcidin knockdown小鼠后可明顯改善小鼠的高鐵負荷狀態(tài),并降低膿毒癥的死亡率。其作用機制與hepcidin調(diào)節(jié)機體鐵代謝的功能密切相關(guān)。
[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.
【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】：2015
【分類號】：R459.7

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