CHOP基因敲除后抑制UUO诱导的小鼠肾脏纤维化的机制研究

发布时间：2018-03-31 07:11

本文选题：UUO　切入点：肾脏纤维化　出处：《华中科技大学》2016年博士论文

【摘要】：研究背景肾脏纤维化是各种慢性肾脏病终末期共同的病理改变,主要的病理特征是肾小球硬化和肾间质纤维化。肾脏纤维化的产生是由于细胞外基质过度沉积,超出了肾脏的清除能力,最终造成肾病终末期(ESRD)不可逆的病理改变。目前并没有针对肾脏纤维化有效的治疗方法,ESRD患者只能依靠终身透析和肾脏移植维持生命。有大量充分的证据表明肾脏系膜细胞和成纤维细胞的活化、肾小管上皮细胞-间充质转化(EMT)、炎症细胞(单核细胞、巨噬细胞和T淋巴细胞)的浸润以及细胞的凋亡是引起肾脏纤维化重要的细胞机制。TGFβ及下游信号调节蛋白Smad的活化很早之前就已经被证实是促进肾脏纤维化形成最重要的分子调节机制,但是肾脏纤维化形成相关的细胞活化机制以及TGFβ表达的诱发因素并没有完全阐述清楚。内质网(ER)是细胞内参与蛋白质合成、转运和修饰,以及脂质合成和Ca2+储存重要的细胞器。当细胞受到诸如氧化应激,缺血和缺氧等刺激时,内质网稳态遭到破坏,影响蛋白质的折叠,并最终引起内质网应激(ER stress)的发生,持续存在而无法及时得到处理的内质网应激会诱导细胞的程序性死亡,即凋亡。大量文献证实,内质网应激相关的细胞凋亡是机体遭受损伤后器官和组织重构的影响因素。例如,在心肌纤维化和肝脏纤维化形成过程中,内质网应激被证实发挥了关键的调节作用。然而,内质网应激在肾脏纤维化病理过程的具体调节机制并未有研究进行阐述。目前仅有Chiang等人利用大鼠肾脏纤维化的模型研究证实为内质网应激确实是肾脏纤维化形成的促进因素,并利用内质网应激抑制剂证实肾脏细胞的凋亡程度与肾脏纤维化的程度是成正比的。综合上述结论,我们首次在慢性肾脏病患者肾脏纤维化形成过程中进行了内质网应激的研究,并利用C/EBP同源蛋白质(Chop)表达基因敲除小鼠的单侧输尿管结扎(UUO)模型分析了凋亡在肾脏纤维化中所发挥的细胞机制和分子机制。我们发现Chop蛋白质表达缺失后对UUO诱导的小鼠肾脏纤维化具有显著的保护作用。由于Chop表达的缺失,在很大程度上避免了肾脏细胞的凋亡和继发性死亡,从而减少了由死亡引起的高迁移率蛋白1 (Hmgbl)的被动释放。与此同时,我们的研究结果还显示Chop基因敲除可以抑制巨噬细胞主动分泌Hmgb1。由此所产生的效应是小鼠肾脏组织中细胞外的Hmgb1总量下降。主动分泌或被动释放到细胞外的Hmgbl与细胞膜表面的Toll样受体4(TLR4)结合,激活MvD88-NF κ B信号通路,最终促进IL-1β的表达。高表达的IL-1β通过促使Tgf β/Smad2/3和Pi3k/Akt信号通路的活化,促进肾脏纤维化的形成。因此,Chop基因敲除后Hmgbl下游整条促进先纤维化形成的信号通路被抑制了。研究方法和结果前期,我们收集了慢性肾脏病终末期患者的肾脏活检组织。通过组织病理学染色,我们观察到终末期肾病患者的肾间质纤维化明显,巨噬细胞浸润显著增加,与此同时,CHOP RT-PCR的检查结果显示患者CHOP mRNA的水平明显高于正常人。为进一步揭示CHOP在肾脏纤维化中的影响和机制,我们建立了Chop基因敲除小鼠模型。单侧输尿管结扎手术(UUO)是诱导动物肾脏纤维化的经典模型。所以我们采用UUO手术分别诱导Chop-/-小鼠和C57BL/6小鼠的肾脏纤维化。在UUO术后14天,通过病理学的检测,我们观察到C57BL/6小鼠肾脏发生了严重的纤维化,肾间质中有大量炎症细胞浸润,肾小管扩张程度及蛋白质管型明显,肾小管刷状缘几乎未见。ER stress相关因子蛋白质及mRNA表达水平的检测结果表明肾脏纤维化病理进程伴随ER stress的发生。然而Chop基因敲除后可以明显改善小鼠UUO术后的所造成的表型。形态学分析结果显示,在相同手术过程处理下,Chop-/-小鼠的肾脏肾盂积水较C57BL/6小鼠明显减少,肾脏皮质厚度改变程度下降。与形态学的表型一致,Chop基因敲除后在极大程度上抑制了小鼠术后肾脏纤维化的发生,并减少了肾间质炎症细胞的浸润,改善了肾小管的扩张程度,保存了肾小管刷状缘的部分完整性。F4/80是小鼠巨噬细胞特异性表面标志,我们通过组织化学染色发现Chop-/-小鼠肾脏间质中巨噬细胞的浸润数目显著下降。同时我们发现CHOP蛋白表达缺失在mRNA和蛋白质水平均阻碍了纤维化相关指标的表达。虽然Chop处于ER stress调控通路的下游,但是可能由于正反馈机制的存在,ER stress相关蛋白的检测结果显示Chop基因敲除后ER stress信号通路上游因子的表达被抑制了。由ER stress诱导表达的Chop蛋白主要功能是调节细胞的程序性死亡(凋亡),但如果凋亡的细胞数目超出了肾脏自身清除能力,细胞会发生继发性死亡。死亡后的细胞被动释放Hmgb1。TUNEL是一项特异性高、敏感性好、表型直观、操作简便的检测细胞凋亡的技术。因此我们首先采用TUNEL检测UUO术后小鼠肾脏凋亡细胞的数目,数据分析结果显示Chop基因敲除后,肾脏凋亡细胞的数目明显减少。随后我们利用免疫印迹和RT-PCR技术检测了凋亡相关蛋白的表达,与TUNEL结果一致,Chop基因的敲除抑制凋亡的发生。乳酸脱氢酶(LDH)是细胞死亡后释放的酶体,其含量可以指示细胞死亡的情况。肾脏溶解物中LDH的检测结果提示Chop-/-小鼠死亡细胞数目减少,由此可以推测Chop蛋白表达的缺失阻碍了细胞的继发性死亡。细胞外Hmgbl的产生方式包括被动释放和主动分泌,通过肾脏组织Hmgbl的免疫组织化学染色和体外细胞培养实验,我们发现两种方式产生的Hmgbl在Chop-/-小鼠中均减少。同时我们证实肾病终末期患者的肾脏同样有大量HMGB1的释放。Toll样受体2(TLR2)、Toll样受体4(TLR4)和RAGE为Hmgb细胞膜上特异性受体。免疫印迹检测三种受体UUO术后在小鼠肾脏组织中的变化,检测结果显示TLR2和RAGE在UUO术后表达升高,但是Chop-/-小鼠和C57BL/6小鼠肾脏中两者的表达并无统计学差异。而TLR4的表达在Chop基因敲除小鼠中受到抑制。TLR4通路下游调节因子MyD88/NF κ B的表达与TLR4一致,在Chop敲除后下降。分泌到细胞外的Hmgb 1可刺激巨噬细胞分泌白介素1β(IL-1β),我们通过免疫印迹和ELISA检测到IL-1β前体和IL-1β的表达下降。IL1β可通过直接和间接两种方式促进纤维化的形成。我们发现两条通路相关的调节因子,在Chop基因敲除后表达量均下降。结论在本篇论文中,我们以ER stress为切入点,Chop为焦点进行肾脏纤维形成机制的研究。在肾病终末期患者的肾脏组织中,我们检测到ER stress相关蛋白表达量的增加。为进一步阐述肾脏纤维化病理机制,我们建立了小鼠肾脏纤维化模型。在研究中,我们证实Chop基因敲除后通过阻碍肾脏细胞的凋亡和继发性坏死,抑制Hmgbl的被动释放和主动分泌,进而抑制了Hmgbl/TLR4/MyD88/NFK B信号通路的激活。由于NFκB的活性被抑制,受其调节的IL1β的表达量下降,最终下调与纤维化形成密切相关的Tgf β/Smad2/3和Pi3k/Akt信号通路,抑制肾脏纤维化的形成。
[Abstract]:The research background of renal fibrosis is the pathological change of chronic end-stage renal disease in common, the major pathological features are glomerular sclerosis and renal interstitial fibrosis. Renal fibrosis is caused by excessive deposition of extracellular matrix, beyond the renal clearance ability, eventually leading to end-stage kidney disease (ESRD). The pathological changes of irreversible and no effective treatment for renal fibrosis, ESRD patients rely on lifelong dialysis and kidney transplant. There is plenty of evidence that activation of fibroblast and mesangial cells, renal tubular epithelial mesenchymal transition (EMT), inflammatory cells (monocytes, macrophages and T lymphocytes). Infiltration and cell apoptosis induced by activated protein Smad is an important mechanism of renal fibrosis in.TGF cells and beta downstream signal had been confirmed to promote Renal fibrosis molecular regulation mechanism of the most important, but the formation of renal fibrosis related cell activation and induce expression of TGF factor is not fully elucidated. The endoplasmic reticulum (ER) is involved in intracellular transport and protein synthesis, modification, and lipid synthesis and storage of Ca2+ cells when cells were as important. Oxidative stress, ischemia and hypoxia, endoplasmic reticulum homeostasis destruction, affect protein folding and cause endoplasmic reticulum stress (ER stress) the occurrence of persistent and cannot receive timely treatment procedures of the endoplasmic reticulum stress may induce cell death, or apoptosis. Several studies have demonstrated that apoptosis, endoplasmic reticulum stress is related to the factors affecting the body subjected to organ and tissue remodeling after injury. For example, in the process of liver fibrosis and myocardial fibrosis, endoplasmic reticulum stress was confirmed. Play a key role. However, there is no study of endoplasmic reticulum stress described in the specific regulation mechanism of renal fibrosis pathological process. At present, only Chiang et al use model of renal fibrosis in rats was confirmed by endoplasmic reticulum stress is the formation of renal fibrosis promoting factors, the degree of apoptosis and the endoplasmic reticulum stress inhibitor confirmed renal cell and is proportional to the degree of renal fibrosis. All the results above, we first studied the formation process of endoplasmic reticulum stress in patients with chronic kidney disease and renal fibrosis, and homologous protein (Chop) expression in C/EBP gene knockout mice with unilateral ureteral obstruction (UUO) model to analyze the cellular mechanisms and molecular mechanisms the apoptosis in renal fibrosis. We found that the loss of Chop protein expression on UUO induced renal fibrosis in mice significantly The protective effect of the Chop. Due to the lack of expression, largely avoided the renal cell apoptosis and secondary death, thereby reducing the caused by death of high mobility protein 1 (Hmgbl) of the passive release. At the same time, our results also showed that Chop gene knockout can inhibit the secretion of macrophage active effect Hmgb1. the resulting is the amount of Hmgb1 cells in the mouse kidney tissue decreased. Toll like receptor Hmgbl and cell membrane surface active or passive secretion release into the extracellular binding 4 (TLR4), the activation of MvD88-NF B signaling pathway, and ultimately promote the expression of IL-1 beta. The high expression of IL-1 beta promotes the activation of Tgf beta /Smad2/3 and Pi3k/Akt signaling pathway, promoting the formation of kidney fibrosis. Therefore, Chop gene knockout Hmgbl downstream signaling pathway to promote the formation of fibrosis were inhibited. The research methods and results before Period, we collected the chronic kidney disease in patients with end-stage renal biopsy. Through histopathological staining, we observed in patients with end-stage renal disease renal interstitial fibrosis, increased macrophage infiltration at the same time, CHOP RT-PCR examination showed that patients with CHOP mRNA was significantly higher than in normal people. In order to further reveal the influence of CHOP in renal fibrosis and mechanism, we established the Chop gene knockout mice model. Unilateral ureteral ligation (UUO) is a classical animal model induced by renal fibrosis. So we use the UUO operation were induced in Chop-/- mice and C57BL/6 mice kidney fibrosis. In the 14 day after UUO, through the detection of pathology. We observed that C57BL/6 mice had severe renal fibrosis, renal interstitial inflammatory cell infiltration, renal tubular dilatation and protein tube significantly, The results of detection of renal tubule brush border almost no.ER stress related factor mRNA and protein expression level showed that renal fibrosis pathological process with the ER stress. However, Chop gene knockout mice can significantly improve the postoperative UUO caused by phenotype. Morphological analysis showed that in the same surgical procedure under the treatment of Chop-/- mice kidney hydronephrosis compared with C57BL/6 mice significantly reduced renal cortical thickness changes decreased. Morphology and phenotype consistent, Chop gene knockout in largely inhibited mice after renal fibrosis occurred, and reduce the infiltration of inflammatory cells in the kidney of quality, improve the degree of renal tubular expansion, part of integrity.F4/80 saved the tubular brush border is mouse macrophage specific surface markers, we found by histochemical staining of Chop-/- mouse kidney interstitial macrophages The number of infiltrating cells decreased significantly. At the same time, we found that the lack of CHOP protein expression in mRNA and protein levels were blocked the expression indexes related to fibrosis. Although the Chop in the ER stress pathway downstream, but may be due to the existence of a positive feedback mechanism, the detection of ER stress protein showed Chop gene inhibited the expression of ER on stress signaling upstream factors in Chop protein. The main function of ER induced by stress expression is the Procedural Regulation of cell death (apoptosis), but if the number of apoptotic cells beyond the ability to remove kidney cells, the occurrence of secondary cell death. After the death of the passive release of Hmgb1.TUNEL is a high specificity and sensitivity well, the phenotype of simple and intuitive, the detection of apoptosis. So we used TUNEL cell apoptosis in mouse kidney number detection after UUO, according to the number of The analysis results show that Chop gene knockout, the number of apoptotic cells in the kidney was significantly reduced. Then we use immunohistochemistry and RT-PCR technique to detect the expression of apoptosis related protein, consistent with the results of TUNEL, Chop gene knockout of the inhibition of apoptosis. Lactate dehydrogenase (LDH) enzyme release after cell death, its content can direct cell death. Detection of renal LDH lysis results suggest that the number of dead cells in Chop-/- mice reduced, it can be speculated that the lack of Chop protein expression prevents secondary cell death. Extracellular Hmgbl production including passive and active release secretion culture experiments by immunohistochemical staining and in vitro kidney cells Hmgbl, we found two ways to produce Hmgbl were reduced in Chop-/- mice. At the same time, we confirmed that patients with end-stage kidney disease also has a large number of HM The release of.Toll GB1 like receptor 2 (TLR2), Toll like receptor 4 (TLR4) and RAGE Hmgb specific membrane receptors. Western blotting after UUO three receptors in the mouse kidney changes, test results showed that the expression of TLR2 and RAGE in UUO increased after operation, but there was no statistical difference both Chop-/- mice and C57BL/6 in mouse kidney. The expression of TLR4 in Chop gene knockout by inhibiting the.TLR4 pathway downstream factor kappa MyD88/NF B expression and TLR4 in mice, Chop knockout decreased. Secreted into the extracellular Hmgb 1 can stimulate macrophage secretion of interleukin 1 beta (we through the expression of IL-1 beta), Western blotting and ELISA detection of IL-1 beta precursor and IL-1 beta decreased.IL1 beta can promote fibrosis through direct and indirect ways. We found two regulatory factor two pathway related, in Chop gene knockout expression were Conclusion decreased. In this thesis, we use ER stress as the starting point, the study of Chop for focus formation mechanism of kidney fiber. In end-stage renal disease in the kidney tissue, we detected ER stress related protein expression increased. In order to further elaborate the pathological mechanism of renal fibrosis, we established mouse kidney fibrosis model. In this study, we confirmed that the Chop gene on apoptosis and secondary necrosis by hindering kidney cells after passive, inhibiting the release of Hmgbl and active secretion, thereby inhibiting the activation of Hmgbl/TLR4/MyD88/NFK B signaling pathway. The NF kappa B activity was inhibited by the expression regulation of IL1 beta decline, eventually downregulation of /Smad2/3 is closely related to the formation of Tgf beta and Pi3k/Akt signaling pathway and inhibit the formation of fibrosis, renal fibrosis.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R692

【相似文献】