本文選題：HNF1b　切入點：ROS　出處：《第四軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：【背景】肝細(xì)胞核因子1b(Hepatocyte nuclear factor 1b,HNF1b)在肝臟、胰腺等重要臟器的發(fā)育階段必不可少,不同位點的突變會導(dǎo)致多個臟器功能紊亂,是近年來生命科學(xué)領(lǐng)域的研究熱點。活性氧(reactive oxygen species,ROS)在2型糖尿病(Type 2diabetes,T2DM)及其并發(fā)癥的發(fā)病機(jī)制中具有重要作用,作為一種信號分子,與肝臟的胚胎發(fā)育密切相關(guān)。既往研究表明,HNF1b在某些疾病發(fā)生發(fā)展中的作用可能通過調(diào)節(jié)體內(nèi)ROS水平來實現(xiàn),但其具體機(jī)制尚不清楚。【目的】課題針對HNF1b與氧化應(yīng)激在疾病發(fā)生發(fā)展過程中調(diào)控機(jī)制不明的問題,構(gòu)建代謝紊亂及肝再生模型,觀察HNF1b在代謝性疾病及肝臟相關(guān)疾病發(fā)生發(fā)展中的關(guān)鍵作用,闡明HNF1b與氧化應(yīng)激的相互作用關(guān)系,探索其信號調(diào)控網(wǎng)絡(luò)在相關(guān)疾病發(fā)生發(fā)展過程中的具體作用機(jī)制�！痉椒ā�1.利用PCB-153構(gòu)建代謝紊亂模型小鼠,隨機(jī)分為四組:對照組(Con),高脂飼料組(HFD)、高脂飼料+PCB-153組(HFD+PCB-153)和PCB-153干預(yù)組(PCB-153)。Con組小鼠普通飼料,HFD組和HFD+PCB-153組小鼠飼喂45%高脂飼料。HFD+PCB-153組和PCB-153組小鼠飲用含4 mg/L PCB-153的水。3個月后檢測血糖、血脂等指標(biāo),檢驗代謝紊亂模型是否成功建立。2.通過質(zhì)粒轉(zhuǎn)染技術(shù)、采用廣譜抗氧化劑N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)以及核轉(zhuǎn)錄因子kappa B(Nuclear Factor kappa B,NF-κB)的抑制劑吡咯烷二硫代氨基甲酸酯(pyrrolidine dithiocarbamate,PDTC)等進(jìn)行干預(yù),研究PCB-153引起代謝紊亂的動物模型中HNF1b、ROS以及NF-κB影響代謝紊亂的具體機(jī)制。3.建立小鼠部分肝切除模型,利用Western Blot、Dihydroethidium(DHE)染色等技術(shù)觀察HNF1b在肝再生過程中的作用;通過質(zhì)粒轉(zhuǎn)染、NAC等干預(yù),進(jìn)一步明確HNF1b/ROS/NF-κB信號通路對肝再生的影響及其作用機(jī)理�！窘Y(jié)果】1.單獨給予PCB-153或HDF,引起血糖升高及胰島素抵抗;HFD+PCB-153加重糖代謝異常,提示PCB-153可以誘發(fā)和加重高脂飼料導(dǎo)致的糖代謝紊亂。2.單獨給予PCB-153或HDF,引起甘油三脂升高,脂滴增大或脂肪重量增加、體積升高;HFD+PCB-153脂質(zhì)累積加重,提示PCB-153可以誘發(fā)和加重高脂飼料導(dǎo)致的脂代謝紊亂。3.PCB-153可以使組織和細(xì)胞中的P65、白細(xì)胞介素-1ɑ(interleukin-1ɑ,IL-1ɑ)、白細(xì)胞介素-6(interleukin-1ɑ,IL-6)mRNA表達(dá)水平升高,給予NF-κB抑制劑PDTC可以抑制PCB-153引發(fā)的IL-1ɑ升高、血脂升高和胰島素抵抗,提示NF-κB介導(dǎo)的炎癥反應(yīng)參與了PCB-153引發(fā)的糖脂代謝紊亂。4.PCB-153促使ROS水平升高、HNF1b和抗氧化酶GPX1的mRNA表達(dá)水平降低。通過外源性NAC干預(yù),可以抑制PCB-153引起的ROS水平升高、P65的mRNA表達(dá)水平升高、甘油三脂升高及胰島素抵抗發(fā)生,過表達(dá)HNF1b與外源性NAC的干預(yù)作用類似,提示PCB-153介導(dǎo)的炎癥反應(yīng)、脂質(zhì)累積和胰島素抵抗同HNF1b表達(dá)異常引起的ROS調(diào)節(jié)失控有關(guān)。5.在70%肝切除模型中,術(shù)后隨著時間的增加,HNF1b、ROS呈現(xiàn)先升高后降低的趨勢,與反應(yīng)肝臟再生修復(fù)能力的細(xì)胞核增殖抗原(Proliferating Cell Nuclear Antigen,PCNA)蛋白表達(dá)變化趨勢基本一致。這提示HNF1b/ROS在肝再生過程中可能發(fā)揮作用。6.過表達(dá)HNF1b或給予NAC處理,70%肝切除模型術(shù)后第一天ROS水平下降,NF-κB、PCNA蛋白表達(dá)下降,提示此時ROS可能作為信號分子,降低ROS水平影響肝再生的正常啟動,不利于肝臟再生修復(fù)�！窘Y(jié)論】1.PCB-153可以誘發(fā)或加重糖脂代謝紊亂,其機(jī)制可能是通過下調(diào)HNF1b的表達(dá)來增加ROS水平,進(jìn)而增強NF-κB介導(dǎo)的炎癥反應(yīng),從而導(dǎo)致脂質(zhì)累積和糖代謝異常。2.HNF1b/ROS/NF-κB信號通路在肝再生過程中可能發(fā)揮作用,過表達(dá)HNF1或下調(diào)ROS、NF-κB水平,影響肝再生的正常啟動,不利于肝臟再生修復(fù)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R575
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本文編號：1601434