钙化性纳米微粒通过ROS-JNK信号通路介导细胞的自噬和凋亡

发布时间：2018-04-15 18:50

  本文选题：钙化性纳米微粒 + 肾小管上皮细胞　； 参考：《广西医科大学》2017年博士论文

【摘要】：目的:钙化性纳米微粒(calcifying nanoparticles,CNPs)在肾结石形成过程中起到重要的作用,但其确切的作用机制目前仍不清楚。本文旨在探讨草酸钙肾结石患者中段尿中培养的CNPs对肾小管上皮细胞的损伤作用及其机制,从而了解CNPs在肾结石形成过程中起到的作用。方法:收集草酸钙肾结石患者(n=20)及健康人(n=10)中段尿进行体外培养并分离CNPs;应用透射电子显微镜(transmission electron microscopy TEM)、扫描电子显微镜(scanning electron microscopy SEM)分析CNPs的形态特征及其内部结构,X-射线能谱分析(energy-dispersive X-ray microanalysis EDX)分析CNPs的主要元素组成,免疫印迹法(Western blotting WB)检测CNPs中胎球蛋白A的表达情况,通过电泳和布朗运动视频分析观察培养的CNPs的粒径分布及其电位情况。通过透射电镜观察草酸钙结石患者肾乳头钙化斑中CNPs存在情况。CNPs(2 MCF)与肾小管上皮细胞(HK-2)分别共培养0h,12h及72h后通过CCK-8(The Cell Counting Kit-8)检测细胞活性,并用流式细胞仪检测活性氧(Reactive oxygen species ROS)的表达情况、线粒体膜电位(Mitochondrial membrane potential MMP)的变化。Ad-mRFP-GFP-LC3双标腺病毒转染后的HK-2与CNPs共培养12h后通过激光共聚焦显微镜观察HK-2的自噬流变化情况。CNPs干预72小时后流式细胞仪测定细胞凋亡率。CNPs干预后通过TEM观察HK-2细胞超微结构变化并观察HK-2的自噬和凋亡现象。WB分析细胞内蛋白水平的变化。结果:1、从草酸钙结石患者的中段尿中可以培养出CNPs,经多种方法鉴定其形态与经典描述一致。CNPs的粒径集中在130nm左右,并检测出CNPs具有负电位。CNPs中包含胎球蛋白-A(Fetuin-A)。在肾乳头钙化斑的超薄切片中可以观察到与培养的CNPs形态具有高度的相似性;2、CCK-8检测结果显示CNPs干预后HK-2的细胞活力随干预的时间增加下降。流式细胞术检测结果提示CNPs可诱导细胞内ROS产生,并检测到HK-2的线粒体膜电位下降。3、我们通过Ad-mRFP-GFP-LC3腺病毒转染实验观察到CNPs干预的早期(12h)HK-2既可以发生自噬现象。流式细胞术检测到CNPs干预72h后细胞出现细胞凋亡情况。细胞超薄切片透射电子显微镜图像显示HK-2可以通过吞噬作用内吞CNPs,细胞内的CNPs包裹于囊泡内,细胞线粒体肿胀,并可以观察到自噬、细胞凋亡及细胞坏死等征象。4、CNPs干预后Bcl-2蛋白的表达下降,而Bax的表达明显升高,自噬指示蛋白LC3-B和Beclin-1表达水平明显增高,通路蛋白p-JNK/JNK表达水平增加(12h)。结论:1、草酸钙肾结石患者的中段尿中可以培养出CNPs,中段尿的培养可以作为泌尿系有无感染CNPs的方法;CNPs可以通过内吞囊泡的方式进入HK-2细胞,CNPs的负电位可能在内吞过程中起到作用;2、CNPs为晶体蛋白复合物,其复制增殖是一种以蛋白为载体的钙磷沉积仿生行为;3、吞噬后的CNPs可通过作用于线粒体诱导细胞内ROS的产生;4、CNPs诱导ROS的过度表达可导致HK-2的自噬与凋亡,严重时还可以引起细胞的坏死;5、CNPs在草酸钙肾结石肾乳头钙斑的形成中起着重要的作用,CNPs的持续钙磷沉积是钙斑中磷酸钙核心形成的基础;6、ROS-JNK通路在CNPs诱导HK-2细胞损伤中起着关键的调控作用。
[Abstract]:Objective: calcifying nanoparticles (calcifying nanoparticles CNPs) plays an important role in the formation of kidney stones, but the exact mechanism is still unclear. This paper aims to explore the cultivation of calcium oxalate kidney stones in patients with urine in CNPs injury and its mechanism of renal tubular epithelial cells, so as to understand the process the role of CNPs in the formation of kidney stones. Methods: Patients with calcium oxalate kidney stones (n=20) and healthy people (n=10) urine were cultured in vitro and separation of CNPs; application of transmission electron microscope (transmission electron microscopy TEM), scanning electron microscopy (scanning electron microscopy SEM) analysis of morphological characteristics and the internal structure of CNPs X-, energy dispersive X-ray analysis (energy-dispersive X-ray microanalysis EDX) the main element analysis CNPs, immunoblotting (Western blotting WB) detection of CNPs in fetal ball The expression of A protein by electrophoresis, and Brown sports video analysis and its potential size distribution observed in cultured CNPs particle. Observed by transmission electron microscopy CNPs in patients with calcium oxalate stone in the presence of calcified plaque in.CNPs (2 MCF) and renal tubular epithelial cells (HK-2) were co cultured with 0h, 12h and 72h by CCK-8 (The Cell Counting Kit-8) to detect cell activity, and active oxygen flow cytometry (Reactive oxygen species ROS) expression, mitochondrial membrane potential (Mitochondrial membrane potential MMP) 12h HK-2 co cultured with the change of CNPs.Ad-mRFP-GFP-LC3 double labeled adenovirus transfection after.CNPs cell apoptosis rate was determined by interference microscopy the changes of.CNPs HK-2 flow of autophagy after 72 hours of treatment by flow cytometry after TEM to observe the change of ultrastructure of HK-2 was observed in HK-2 of the autophagy and apoptosis of confocal laser .WB analysis of the changes of protein levels in cells. Results: 1, from the urine of patients with calcium oxalate stones can develop CNPs, identified by several methods of its form and the classical description of consistent.CNPs particle size at about 130nm, and the detection of CNPs with negative potential.CNPs contains fetuin -A (Fetuin-A) in the renal papilla calcified plaque in thin sections can be observed with cultured CNPs morphology with a high degree of similarity; 2, CCK-8 showed that the HK-2 cell activity with CNPs intervention after the intervention increased decreased. Test results indicate that CNPs induced intracellular ROS production by flow cytometry, and detected the mitochondrial membrane potential decline in HK-2.3, we observed that early CNPs intervention by Ad-mRFP-GFP-LC3 adenovirus transfection experiment (12h) HK-2 can occur autophagy. Flow cytometry detected CNPs intervention after 72h cells apoptosis Cell. Ultrathin section transmission electron microscopy images showed that HK-2 by phagocytosis CNPs endocytosis, intracellular CNPs encapsulated in vesicles, mitochondria swelling, and can be observed in autophagy, apoptosis and cell necrosis and other signs of.4, CNPs after the intervention of the expression of Bcl-2 protein decreased, while the expression of Bax was significantly increased. Autophagy indicates protein LC3-B and Beclin-1 expression level was significantly increased, the expression level of p-JNK/JNK protein pathway increased (12h). Conclusion: 1. Patients with calcium oxalate kidney stone in urine can develop CNPs, cultivate Medistream urine can be used as a method of urinary tract infection of CNPs; CNPs can swallow vesicles into the HK-2 cells, negative potential CNPs may play a role in endocytosis; 2, CNPs crystal protein complex, its replication is a protein carrier for biomimetic deposition of calcium and phosphorus 3, phagocytosis of CNPs; By acting on mitochondria induced by intracellular ROS generation; 4, the over expression of CNPs induced by ROS can induce autophagy and apoptosis of HK-2, serious when still can cause cell necrosis; 5, CNPs plays an important role in the formation of calcium oxalate kidney stones, renal papilla calcification in CNPs sustained calcium phosphate the deposition is the basis of calcium phosphate calcium plaque in the formation of the core; 6, ROS-JNK pathway plays a critical role in the regulation of HK-2 cell injury induced by CNPs.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R692.4

【相似文献】

相关期刊论文 前10条

1 刘培现;老年钙化性主动脉瓣关闭不全6例临床分析[J];武汉医学杂志;1995年02期

2 马景擰,洪国良,于士柱,孔繁昌;罕见钙化性毛细胞型星形细胞瘤一例[J];中华神经外科杂志;2001年05期

3 支莉,李小清;因钙化性二尖瓣狭窄行经皮二尖瓣连合部切开术的晚期效果[J];心血管病学进展;2001年03期

4 史淑范,高晓军,林淑珍,胡卫建;2490名中老年干部钙化性主动脉瓣病的发病情况及超声心动图分析[J];中国超声医学杂志;1990年S1期

5 吴明灿;;前颅窝底巨大慢性钙化性炎性肉芽肿1例[J];湖北省卫生职工医学院学报;1993年02期

6 曲莉,彭昌鼎,于伟琦,邓映和;退行性钙化性心瓣膜疾病的临床与声象图表现[J];前卫医药杂志;1996年03期

7 吴世清;高河元;;慢性钙化性胰腺炎1例[J];福建医药杂志;1988年04期

8 黄启麒;;老年性钙化性主动脉瓣病一例报告[J];中国疗养医学;1993年03期

9 王麟根;张建;;慢性钙化性胰腺炎引起梗阻性黄疸一例报告[J];上海医学;1984年04期

10 陈传宏;钙化性胰腺炎:钙化类型与胰腺影象对照[J];国外医学(临床放射学分册);1985年06期

相关会议论文 前3条

1 闫洁;杨希川;叶庆佾;郝飞;;钙化性纤维性肿瘤1例[A];中华医学会第14次全国皮肤性病学术年会论文汇编[C];2008年

2 裴育;庞萍;王海珍;韩晓菲;王先令;杜锦;杨国庆;吕朝晖;窦京涛;母义明;陆菊明;;胰腺纤维钙化性糖尿病临床特征分析及3例报告[A];中华医学会第十一次全国内分泌学学术会议论文汇编[C];2012年

3 程蕴琳;刘莉;李百周;张小进;;探讨瓣膜钙化性病变与动脉粥样硬化的关系——附一例瓣膜病变临床病理分析[A];中华医学会第九次全国老年医学学术会议暨第三届全国老年动脉硬化与周围血管疾病专题研讨会论文汇编[C];2009年

相关重要报纸文章 前1条

1 记者 谭克扬 通讯员 魏红 吴日明;肝胆等疾病致病之谜有望解开[N];湖南日报;2004年

相关博士学位论文 前1条

1 吴基华;钙化性纳米微粒通过ROS-JNK信号通路介导细胞的自噬和凋亡[D];广西医科大学;2017年

相关硕士学位论文 前2条

1 孟冬冬;钙化性纳米微粒与人肾小管上皮细胞相互作用研究[D];广西医科大学;2011年

2 李伟;去细胞结合光氧化技术处理牛颈静脉带瓣管道抗钙化性能研究[D];中南大学;2007年

，

本文编号：1755345