HNF1b在代谢紊乱及肝再生过程中作用机制的研究

发布时间：2018-03-12 11:52

  本文选题：HNF1b　切入点：ROS　出处：《第四军医大学》2017年博士论文　论文类型：学位论文

【摘要】：【背景】肝细胞核因子1b(Hepatocyte nuclear factor 1b,HNF1b)在肝脏、胰腺等重要脏器的发育阶段必不可少,不同位点的突变会导致多个脏器功能紊乱,是近年来生命科学领域的研究热点。活性氧(reactive oxygen species,ROS)在2型糖尿病(Type 2diabetes,T2DM)及其并发症的发病机制中具有重要作用,作为一种信号分子,与肝脏的胚胎发育密切相关。既往研究表明,HNF1b在某些疾病发生发展中的作用可能通过调节体内ROS水平来实现,但其具体机制尚不清楚。【目的】课题针对HNF1b与氧化应激在疾病发生发展过程中调控机制不明的问题,构建代谢紊乱及肝再生模型,观察HNF1b在代谢性疾病及肝脏相关疾病发生发展中的关键作用,阐明HNF1b与氧化应激的相互作用关系,探索其信号调控网络在相关疾病发生发展过程中的具体作用机制。【方法】1.利用PCB-153构建代谢紊乱模型小鼠,随机分为四组:对照组(Con),高脂饲料组(HFD)、高脂饲料+PCB-153组(HFD+PCB-153)和PCB-153干预组(PCB-153)。Con组小鼠普通饲料,HFD组和HFD+PCB-153组小鼠饲喂45%高脂饲料。HFD+PCB-153组和PCB-153组小鼠饮用含4 mg/L PCB-153的水。3个月后检测血糖、血脂等指标,检验代谢紊乱模型是否成功建立。2.通过质粒转染技术、采用广谱抗氧化剂N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)以及核转录因子kappa B(Nuclear Factor kappa B,NF-κB)的抑制剂吡咯烷二硫代氨基甲酸酯(pyrrolidine dithiocarbamate,PDTC)等进行干预,研究PCB-153引起代谢紊乱的动物模型中HNF1b、ROS以及NF-κB影响代谢紊乱的具体机制。3.建立小鼠部分肝切除模型,利用Western Blot、Dihydroethidium(DHE)染色等技术观察HNF1b在肝再生过程中的作用;通过质粒转染、NAC等干预,进一步明确HNF1b/ROS/NF-κB信号通路对肝再生的影响及其作用机理。【结果】1.单独给予PCB-153或HDF,引起血糖升高及胰岛素抵抗;HFD+PCB-153加重糖代谢异常,提示PCB-153可以诱发和加重高脂饲料导致的糖代谢紊乱。2.单独给予PCB-153或HDF,引起甘油三脂升高,脂滴增大或脂肪重量增加、体积升高;HFD+PCB-153脂质累积加重,提示PCB-153可以诱发和加重高脂饲料导致的脂代谢紊乱。3.PCB-153可以使组织和细胞中的P65、白细胞介素-1ɑ(interleukin-1ɑ,IL-1ɑ)、白细胞介素-6(interleukin-1ɑ,IL-6)mRNA表达水平升高,给予NF-κB抑制剂PDTC可以抑制PCB-153引发的IL-1ɑ升高、血脂升高和胰岛素抵抗,提示NF-κB介导的炎症反应参与了PCB-153引发的糖脂代谢紊乱。4.PCB-153促使ROS水平升高、HNF1b和抗氧化酶GPX1的mRNA表达水平降低。通过外源性NAC干预,可以抑制PCB-153引起的ROS水平升高、P65的mRNA表达水平升高、甘油三脂升高及胰岛素抵抗发生,过表达HNF1b与外源性NAC的干预作用类似,提示PCB-153介导的炎症反应、脂质累积和胰岛素抵抗同HNF1b表达异常引起的ROS调节失控有关。5.在70%肝切除模型中,术后随着时间的增加,HNF1b、ROS呈现先升高后降低的趋势,与反应肝脏再生修复能力的细胞核增殖抗原(Proliferating Cell Nuclear Antigen,PCNA)蛋白表达变化趋势基本一致。这提示HNF1b/ROS在肝再生过程中可能发挥作用。6.过表达HNF1b或给予NAC处理,70%肝切除模型术后第一天ROS水平下降,NF-κB、PCNA蛋白表达下降,提示此时ROS可能作为信号分子,降低ROS水平影响肝再生的正常启动,不利于肝脏再生修复。【结论】1.PCB-153可以诱发或加重糖脂代谢紊乱,其机制可能是通过下调HNF1b的表达来增加ROS水平,进而增强NF-κB介导的炎症反应,从而导致脂质累积和糖代谢异常。2.HNF1b/ROS/NF-κB信号通路在肝再生过程中可能发挥作用,过表达HNF1或下调ROS、NF-κB水平,影响肝再生的正常启动,不利于肝脏再生修复。
[Abstract]:[background] hepatocyte nuclear factor 1B (Hepatocyte nuclear factor 1b, HNF1b) in the liver, pancreas and other important organs essential developmental stages, different mutation sites will lead to multiple organ dysfunction, is a research hotspot in the field of life science in recent years. Reactive oxygen species (reactive oxygen species ROS (Type) in type 2 diabetes mellitus 2diabetes, T2DM) plays an important role in the pathogenesis and its complications, as a signal molecule, is closely related with the liver of embryo development. Previous studies showed that HNF1b in the occurrence and development of as can be achieved by adjusting the level of ROS in certain diseases, but its mechanism is not clear. [Objective] article HNF1b and oxidative stress in the disease process in the regulation mechanism of the problem is unknown, construction of metabolic disorder and liver regeneration model, observe the HNF1b in metabolic diseases and liver diseases A key role in the development of interaction relationship between HNF1b and oxidative stress, explore the specific mechanism of its signal transduction network in the process in the development of related diseases. [Methods] 1. using PCB-153 to construct the model of metabolic disturbance of mice, were randomly divided into four groups: control group (Con), high fat diet group (HFD) high fat diet, +PCB-153 group (HFD+PCB-153) and PCB-153 group (PCB-153) in.Con group with normal diet, blood sugar detection of HFD group and HFD+PCB-153 group of mice fed high-fat diet for 45%.HFD+PCB-153 group and PCB-153 group of mice drinking water containing 4 mg/L PCB-153 after.3 months, blood lipid metabolic disorder, test whether the model was successfully established.2. by plasmid transfection technique, using broad-spectrum antioxidant N-acetylcysteine (N- N-acetyl-L-cysteine, NAC) and nuclear transcription factor kappa B (Nuclear Factor kappa B, NF- K B) two inhibitor pyrrolidine dithiocarbamate Formate (pyrrolidine dithiocarbamate, PDTC) and intervention study of PCB-153 induced HNF1b animal model of metabolic disorders, ROS and NF- K B effects of specific mechanisms of metabolic disorder of.3. mouse hepatectomy was established by using Western model, Blot, Dihydroethidium (DHE) staining techniques to study the role of HNF1b in the process of liver regeneration; through plasmid transfection, NAC interference, to further clarify the effect of HNF1b/ROS/NF- B pathway on liver regeneration and its mechanism of action. [results] 1. separate administration of PCB-153 or HDF, caused by elevated blood sugar and insulin resistance; HFD+PCB-153 exacerbation of abnormal glucose metabolism, suggesting that PCB-153 can induce and aggravate.2. glucose metabolism of high fat diet caused by administration of PCB-153 alone or HDF, caused by glycerin three fat increased, lipid droplets increased or fat weight increase, the volume increased; HFD+PCB-153 increased lipid accumulation, suggesting that PCB-153 can induce and Increased.3.PCB-153 lipid metabolic disorder in high fat diet can lead to the tissue and cells in P65, interleukin -1 alpha (interleukin-1 alpha, alpha IL-1), interleukin -6 (interleukin-1 alpha, IL-6) increased the expression level of mRNA, NF- kappa B inhibitor PDTC can inhibit PCB-153 induced IL-1 alpha increased. Elevated blood lipids and insulin resistance, the inflammatory reaction mediated by NF- kappa B in elevated ROS level.4.PCB-153 to lipid metabolic disorder caused by PCB-153, the expression level of HNF1b and antioxidant enzymes of GPX1 mRNA decreased. Through the intervention of exogenous NAC can inhibit the production of PCB-153, increased by ROS level, increased the expression level of P65 mRNA, glycerin three high lipid and insulin resistance, overexpression of HNF1b and the intervention effect of exogenous NAC, suggesting that PCB-153 mediated inflammatory response, lipid accumulation and insulin resistance with the abnormal expression of HNF1b induced by ROS regulation About 70%.5. in the liver, postoperative increase with time, HNF1b, ROS atfirstincreasedandthendecreased, proliferating cell nuclear antigen repair ability and the reaction of liver regeneration (Proliferating Cell Nuclear Antigen, PCNA) expression of the trend is basically the same. The results suggest that HNF1b/ROS may play a role in the over expression of.6. HNF1b or pay NAC in the process of liver regeneration, 70% hepatectomy model after surgery decreased the first day the level of ROS, NF- kappa B, PCNA protein expression decreased, suggesting that the ROS may act as a signal molecule, reduce the level of ROS affect the normal start of liver regeneration, is not conducive to liver regeneration. [Conclusion] 1.PCB-153 can induce or aggravate lipid metabolic disorders and the mechanism is probably through down regulating the expression of HNF1b to increase the ROS level, thus enhancing the inflammatory reaction mediated by NF- kappa B, which leads to abnormal glucose metabolism and lipid accumulation in.2.HNF1b/ROS/NF- Kappa B signaling pathway may play a role in the process of liver regeneration. Overexpression of HNF1 or down regulation of ROS and NF- kappa B level affect the normal start of liver regeneration, which is not conducive to liver regeneration and repair.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575
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本文编号：1601434