Stats激活对缺血/再灌注诱导近曲肾小管上皮细胞凋亡的影响及其细胞内机制
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摘要
背景及目的：急性肾衰竭(Acute renal failure，ARF)是常见的临床综合症。近半个世纪以来，在住院病人、尤其是重症监护病人中，ARF病死率一直居高不下。ARF最主要的形式是急性肾小管坏死(acute tubular necrosis，ATN)，而凋亡是ATN时肾小管上皮细胞死亡的主要方式，是ATN治疗的重要靶点。但目前对引起肾小管上皮细胞凋亡的分子机制尚不清楚。深入了解ATN时凋亡的细胞内机制非常重要。Janus激酶-信号传导与转录活化因子(Januskinase-signal transducers and activators of transcription，Jak-Stat)信号途径可介导炎症、增殖、凋亡、发育、肿瘤等过程，并受细胞因子信号传导抑制因子(suppressors of cytokinesignaling，SOCS)负反馈调控。目前尚不清楚Jak-Stat-SOCS通路在ATN病理生理过程中的作用。本研究旨在探讨Jak-Stat-SOCS信号途径是否参与缺血再灌注诱导ATN中的发病机制，试图明确该途径对缺血再灌注诱导的近曲肾小管上皮细胞凋亡的调控作用，以期寻找阻断上皮细胞凋亡、改善ARF临床预后的有效方法。
方法：制作缺血再灌注肾损伤的细胞模型，诱导体外培养的近曲肾小管上皮细胞凋亡。Western印迹、细胞免疫荧光染色检测Stat1，Stat3激活。使用特异性阻断剂AG 490抑制酪氨酸激酶Jak2，或利用RNA干涉(RNA interference，RNAi)技术分别基因敲低Stat1和Stat3，通过流式细胞术检测其对缺血再灌注诱导近曲肾小管上皮细胞凋亡的影响，判定Stat1和Stat3激活在缺血再灌注诱导肾小管上皮细胞凋亡中的作用。应用反转录-多聚酶链反应(reversetranscription-polymerase chain reaction，RT-PCR)，Western印迹检测Stats负反馈因子SOCS3是否被诱导表达，并利用RNAi技术基因敲低SOCS3，通过Western印迹和流式细胞术观察SOCS3敲低后对缺血再灌注诱导Stat1，Stat3激活和上皮细胞凋亡的影响，探讨Jak2-Stat1／Sta3-SOCS3途径激活在缺血再灌注诱导肾小管上皮细胞凋亡中的作用。最后稳定转染Stat3组成性激活质粒，通过流式细胞术检测Stat3组成性激活质粒过表达对缺血再灌注诱导小管上皮细胞凋亡和线粒体膜电位的影响，以进一步明确Stat3激活的保护作用及机制。
Background and objective Acute renal failure (ARF) remains a common clinical entity and results in unacceptable high mortality among hospitalized patients, especially in the intensive care unit setting. Despite advances in medical care, the outcome has not improved during the past 50 years. Acute renal failure(ATN) is the most common form of ARF and is characterized by tubular cell death. Recent evidence suggests that apoptosis is the primary mode of tubular cell death found in ATN and apoptosis is an important therapeutic target in ATN. Unfortunately, the molecular basis underlying apoptosis during ATN is largely unknown. It is important to understand the intracellular signaling pathways that are involved in apoptosis in ATN. Janus kinase-signal transducers and activators of transcription(Jak-Stat) pathway participates in imflammation, apoptosis, proliferation, tumorigenesis and negatively regulated by the family of suppressors of cytokine signaling ( SOCS ) . To our knowledge , there have been no reports regarding the role of Jak-Stat-SOCS in ATN and their contribution to tubular cell apoptosis. The present study was conducted to examine the role of the Jak-Stat-SOCS pathway in kidney proximal tubular epithelial cell apoptosis following ischemia and reperfusion injury (IRI) , and hopefully, to open a new therapeutic modality to mitigate the development of ATN.
方法：制作缺血再灌注肾损伤的细胞模型，诱导体外培养的近曲肾小管上皮细胞凋亡。Western印迹、细胞免疫荧光染色检测Stat1，Stat3激活。使用特异性阻断剂AG 490抑制酪氨酸激酶Jak2，或利用RNA干涉(RNA interference，RNAi)技术分别基因敲低Stat1和Stat3，通过流式细胞术检测其对缺血再灌注诱导近曲肾小管上皮细胞凋亡的影响，判定Stat1和Stat3激活在缺血再灌注诱导肾小管上皮细胞凋亡中的作用。应用反转录-多聚酶链反应(reversetranscription-polymerase chain reaction，RT-PCR)，Western印迹检测Stats负反馈因子SOCS3是否被诱导表达，并利用RNAi技术基因敲低SOCS3，通过Western印迹和流式细胞术观察SOCS3敲低后对缺血再灌注诱导Stat1，Stat3激活和上皮细胞凋亡的影响，探讨Jak2-Stat1／Sta3-SOCS3途径激活在缺血再灌注诱导肾小管上皮细胞凋亡中的作用。最后稳定转染Stat3组成性激活质粒，通过流式细胞术检测Stat3组成性激活质粒过表达对缺血再灌注诱导小管上皮细胞凋亡和线粒体膜电位的影响，以进一步明确Stat3激活的保护作用及机制。
Background and objective Acute renal failure (ARF) remains a common clinical entity and results in unacceptable high mortality among hospitalized patients, especially in the intensive care unit setting. Despite advances in medical care, the outcome has not improved during the past 50 years. Acute renal failure(ATN) is the most common form of ARF and is characterized by tubular cell death. Recent evidence suggests that apoptosis is the primary mode of tubular cell death found in ATN and apoptosis is an important therapeutic target in ATN. Unfortunately, the molecular basis underlying apoptosis during ATN is largely unknown. It is important to understand the intracellular signaling pathways that are involved in apoptosis in ATN. Janus kinase-signal transducers and activators of transcription(Jak-Stat) pathway participates in imflammation, apoptosis, proliferation, tumorigenesis and negatively regulated by the family of suppressors of cytokine signaling ( SOCS ) . To our knowledge , there have been no reports regarding the role of Jak-Stat-SOCS in ATN and their contribution to tubular cell apoptosis. The present study was conducted to examine the role of the Jak-Stat-SOCS pathway in kidney proximal tubular epithelial cell apoptosis following ischemia and reperfusion injury (IRI) , and hopefully, to open a new therapeutic modality to mitigate the development of ATN.
引文
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