糖原合成酶激酶3β调节足细胞骨架在足细胞损伤机制及治疗中的作用

发布时间：2018-04-27 19:59

本文选题：足细胞 + 细胞骨架　；参考：《南京大学》2014年博士论文

【摘要】：肾小球疾病是中国终末期肾病最主要的原发病。足细胞作为肾小球滤过屏障的最外层,对维持正常肾小球滤过起着极其重要的作用。足细胞损伤被认为是蛋白尿产生的主要原因之一。对于足细胞损伤机制的研究及相对应治疗靶点的探索因此极为重要。足细胞损伤以足突融合、足细胞脱落为主要表现。足突的融合与细胞骨架中的微丝结构改变密切相关,而足细胞脱落后细胞的代偿取决于由微管蛋白聚合与解聚调控的初级突起的延伸。本研究以阿霉素在体内和体外诱导足细胞损伤,分别探讨微丝及微管在细胞损伤、修复中的作用；证明了糖原合成酶激酶3β (Glycogen synthase kinase 3β, GSK3β)在足细胞中一方面可通过调节黏着斑(Focal adhesion, FA)组成蛋白桩蛋白(paxillin)的活性,改变黏着斑稳定性,从而影响微丝结构,另一方面可通过调节微管相关蛋白(microtubule associate protein, MAP)的活性,影响微管的聚合与解聚以及足细胞初级突起的延伸,控制着足细胞损伤后的代偿效率。桩蛋白的磷酸化可增加黏着斑的转变速率,从而增加细胞活动力,促进微丝解聚。当体外培养的足细胞受到损伤时,出现微丝的解聚,黏着斑数量增多,面积减小及细胞活动力的增加,同时伴随应力纤维(stress fiber)的解聚,细胞形态的萎缩。这些表现伴随着桩蛋白磷酸化的增加以及GSK3P的过度活化。氯化锂(Lithium chloride,LiC1),一种高度认可的情绪稳定剂在阿霉素损伤前预处理可预防GSK3β的过度活化,减少桩蛋白磷酸化,降低黏着斑的转变速率,阻止了足细胞活动力的过度升高。另外,通过转染质粒使足细胞表达GSK3β不同突变种可以导致不同的细胞表现,且与锂剂或阿霉素处理效果相似。细胞表达酶失活的GSK3β与锂剂类似,而表达持续激活S9A位点的GSK3p则与阿霉素处理类似,且表达酶失活的GSK3β的细胞可以对抗阿霉素的损伤。这表明,锂剂对足细胞的保护作用在较大程度上是通过对GSK3β活性的抑制。同时,桩蛋白也被证明是GSK3β的下游蛋白。而当体内足细胞受阿霉素损伤后也出现GSK3β活性增高,桩蛋白磷酸化增加,足细胞中微丝减少,足突融合；提前给予小鼠氯化锂可降低GSK3β活性,同时使桩蛋白的磷酸化降低,足突中微丝的解聚减少,足突融合及肾小球硬化降低。本研究证明锂剂抑制了阿霉素导致的足细胞中GSK3β过度活化,桩蛋白过磷酸化,维持微丝结构,减轻足突融合。综上,GSK3β调节的黏着斑的动力可能是足细胞损伤的一个可能机理,而小剂量氯化锂可能为预防足细胞损伤提供了一个新思路。当足细胞损伤脱落后,残余的足细胞可能与神经细胞轴突损伤后延长机制类似,通过延伸初级突起发生代偿,企图掩盖裸露的基底膜。微管作为足细胞初级突起中的主要骨架结构,在足细胞形态维持和初级突起生成、延长和分支中起重要作用。本研究探讨了两种重要的微管相关蛋白(microtubule associate protein, MAP) tau蛋白和坍塌反应调节蛋白(collapsin response mediator protein, CRMP)2作为GSK3β下游蛋白,在足细胞中的作用。由阿霉素或表达持续激活S9A位点引起的GSK3β过度活化均导致tau蛋白和CRMP2的过磷酸化、微管的解聚及细胞形态的萎缩、细胞突起的缩短。锂剂或表达酶失活的GSK3β在足细胞中可逆转以上改变。在阿霉素模型中,小剂量单次氯化锂治疗在无明显改善足细胞丢失的情况下减少了蛋白尿和肾小球硬化。进一步的机制研究表明,锂剂降低了GSK3β活性,减少了tau和CRMP2的磷酸化,增加了微管的聚集和稳定性,促进了足细胞初级突起的延伸,从而改善足细胞的代偿。因此,针对GSK3β调节的足细胞微管延伸和足细胞丢失后的代偿可能为足细胞损伤带来新的治疗方向。综上所述,本研究阐述了GSK3β在足细胞损伤中促进足细胞黏着斑转变增快、微丝及微管解聚,与足突融合相关,可能为足细胞损伤机制；氯化锂抑制GSK3β活性可通过防止微丝解聚、足突融合从而减轻足细胞损伤,对预防肾移植复发有潜在意义；氯化锂后处理可通过促进残余足细胞初级突起延伸,增加代偿而促进修复,为足细胞损伤疾病的治疗提供了可能的新方向。
[Abstract]:Glomerular disease is the primary primary disease of end-stage renal disease in China. Podocyte, the outermost layer of the glomerular filtration barrier, plays an important role in maintaining normal glomerular filtration. Podocyte injury is considered as one of the main causes of proteinuria. Research on the mechanism of foot cell damage and the target of relative treatment The fusion of podocytes is the main manifestation of podocyte fusion. Foot process fusion is closely related to the changes of microfilament structure in the cytoskeleton, and the compensation of the cells after the peeling of the podocytes depends on the extension of the primary protuberance regulated by microtubulin polymerization and depolymerization. The effect of microfilament and microtubule on cell damage and repair is discussed respectively. It is proved that the glycogen synthetase kinase 3 beta (Glycogen synthase kinase 3 beta, GSK3 beta) can make up the activity of protein pile protein (paxillin) by regulating the adhesion plaque (Focal adhesion, FA) in the podocyte, and change the stability of the sticky spot, so as to change the stability of the plaque. The structure of the microfilament, on the other hand, can be controlled by the regulation of the activity of microtubule associate protein (MAP), the polymerization and depolymerization of microtubules, and the extension of the primary protuberance of the podocyte. The phosphorylation of PCR can increase the rate of change of the plaque and increase the activity of the cell. To promote the depolymerization of microfilament. When the cultured podocytes are damaged, the depolymerization of microfilament, the increase of the number of plaque, the decrease of the area and the increase of cell activity, and the depolymerization of the stress fiber (stress fiber) and the atrophy of the cell morphology are accompanied by the increase of phosphorylation of piled protein and the overactivation of GSK3P. Lithium (Lithium chloride, LiC1), a highly recognized emotional stabilizer preconditioning before adriamycin damage can prevent the overactivation of GSK3 beta, reduce the phosphorylation of PCR, reduce the rate of change in the plaque, and prevent the excessive increase in the activity of the podtlet. In addition, the expression of GSK3 beta variant can lead to the expression of GSK3 beta mutants by the transfer plasmids. The cell expression of different cells is similar to that of lithium or adriamycin. The GSK3 beta that expresses the inactivation of the enzyme is similar to that of the lithium agent, while the GSK3p expressing the S9A site is similar to the adriamycin treatment, and the cells expressing the inactivated GSK3 beta can antagonism the damage of adriamycin. This indicates that the protective effect of the lithium agent on the foot cells is in Jiao Dacheng. On the degree of inhibition of GSK3 beta activity, PCR is also proved to be a downstream protein of GSK3 beta, and when adriamycin is damaged in the body, the activity of GSK3 beta is increased, the phosphorylation of Paxin increases, the microfilament in the podocytes decreases, and the foot process is fused. The decrease of phosphorylation, depolymerization of microfilament in the poddal process, the fusion of poddate and the decrease of glomerulosclerosis. This study demonstrated that lithium inhibits the overactivation of GSK3 beta in adriamycin induced podocytes, Posin perphosphorylation, microfilament structure, and amalgamation of poddate. In conclusion, the power of GSK3 beta regulated plaque may be one of the potential of foot cell injury. A small dose of lithium chloride may provide a new idea for the prevention of foot cell injury. When the foot cell injury falls off, the remnant Poddar may be similar to the extension mechanism after the nerve cell axon damage. It can cover the bare basement membrane by extending the primary protuberance to cover the bare basement membrane. Microtubule is the main main protuberance in the Poda. In this study, two important microtubule related proteins (microtubule associate protein, MAP) tau protein and collapse response regulation protein (collapsin response mediator protein, CRMP) 2 were considered as GSK3 beta downstream proteins in the foot thin. The action in the cell. Excessive activation of GSK3 beta caused by Adriamycin or the expression of continuous activation of the S9A locus all lead to phosphorylation of tau protein and CRMP2, the depolymerization of microtubules and the atrophy of cell morphology, and the shortened process of cell protuberance. The lithium or GSK3 beta inactivated GSK3 beta can be reversed in the Poda. In the adriamycin model, small dose single chlorine Lithium-ion therapy reduces proteinuria and glomerulosclerosis without significantly improving podocyte loss. Further mechanism studies show that lithium reduces the GSK3 beta activity, reduces the phosphorylation of tau and CRMP2, increases the aggregation and stability of microtubules, promotes the extension of the primary protrusions of the podocytes, and thus improves the compensatory effect of the podocyte. In this case, GSK3 beta regulated foot cell microtubule extension and foot cell loss may bring new therapeutic direction for podocyte injury. To sum up, this study explained that GSK3 beta in podocyte injury promotes the rapid growth of podocyte adhesion, microfilament and microtubule disaggregation, and is associated with foot process fusion, which may be the mechanism of podocyte injury. Lithium chloride inhibits GSK3 beta activity by preventing the depolymerization of microfilament, foot process fusion and alleviating foot cell damage, which is of potential significance for preventing the recurrence of renal transplantation. Lithium chloride treatment can provide a possible new direction for the treatment of foot cell injury by promoting the extension of the primary protuberance of the residual podocyte, increasing compensation and promoting the repair.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

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