本文選題：鈣化性納米微粒 + 腎小管上皮細(xì)胞��； 參考：《廣西醫(yī)科大學(xué)》2017年博士論文

【摘要】：目的:鈣化性納米微粒(calcifying nanoparticles,CNPs)在腎結(jié)石形成過(guò)程中起到重要的作用,但其確切的作用機(jī)制目前仍不清楚。本文旨在探討草酸鈣腎結(jié)石患者中段尿中培養(yǎng)的CNPs對(duì)腎小管上皮細(xì)胞的損傷作用及其機(jī)制,從而了解CNPs在腎結(jié)石形成過(guò)程中起到的作用。方法:收集草酸鈣腎結(jié)石患者(n=20)及健康人(n=10)中段尿進(jìn)行體外培養(yǎng)并分離CNPs;應(yīng)用透射電子顯微鏡(transmission electron microscopy TEM)、掃描電子顯微鏡(scanning electron microscopy SEM)分析CNPs的形態(tài)特征及其內(nèi)部結(jié)構(gòu),X-射線能譜分析(energy-dispersive X-ray microanalysis EDX)分析CNPs的主要元素組成,免疫印跡法(Western blotting WB)檢測(cè)CNPs中胎球蛋白A的表達(dá)情況,通過(guò)電泳和布朗運(yùn)動(dòng)視頻分析觀察培養(yǎng)的CNPs的粒徑分布及其電位情況。通過(guò)透射電鏡觀察草酸鈣結(jié)石患者腎乳頭鈣化斑中CNPs存在情況。CNPs(2 MCF)與腎小管上皮細(xì)胞(HK-2)分別共培養(yǎng)0h,12h及72h后通過(guò)CCK-8(The Cell Counting Kit-8)檢測(cè)細(xì)胞活性,并用流式細(xì)胞儀檢測(cè)活性氧(Reactive oxygen species ROS)的表達(dá)情況、線粒體膜電位(Mitochondrial membrane potential MMP)的變化。Ad-mRFP-GFP-LC3雙標(biāo)腺病毒轉(zhuǎn)染后的HK-2與CNPs共培養(yǎng)12h后通過(guò)激光共聚焦顯微鏡觀察HK-2的自噬流變化情況。CNPs干預(yù)72小時(shí)后流式細(xì)胞儀測(cè)定細(xì)胞凋亡率。CNPs干預(yù)后通過(guò)TEM觀察HK-2細(xì)胞超微結(jié)構(gòu)變化并觀察HK-2的自噬和凋亡現(xiàn)象。WB分析細(xì)胞內(nèi)蛋白水平的變化。結(jié)果:1、從草酸鈣結(jié)石患者的中段尿中可以培養(yǎng)出CNPs,經(jīng)多種方法鑒定其形態(tài)與經(jīng)典描述一致。CNPs的粒徑集中在130nm左右,并檢測(cè)出CNPs具有負(fù)電位。CNPs中包含胎球蛋白-A(Fetuin-A)。在腎乳頭鈣化斑的超薄切片中可以觀察到與培養(yǎng)的CNPs形態(tài)具有高度的相似性;2、CCK-8檢測(cè)結(jié)果顯示CNPs干預(yù)后HK-2的細(xì)胞活力隨干預(yù)的時(shí)間增加下降。流式細(xì)胞術(shù)檢測(cè)結(jié)果提示CNPs可誘導(dǎo)細(xì)胞內(nèi)ROS產(chǎn)生,并檢測(cè)到HK-2的線粒體膜電位下降。3、我們通過(guò)Ad-mRFP-GFP-LC3腺病毒轉(zhuǎn)染實(shí)驗(yàn)觀察到CNPs干預(yù)的早期(12h)HK-2既可以發(fā)生自噬現(xiàn)象。流式細(xì)胞術(shù)檢測(cè)到CNPs干預(yù)72h后細(xì)胞出現(xiàn)細(xì)胞凋亡情況。細(xì)胞超薄切片透射電子顯微鏡圖像顯示HK-2可以通過(guò)吞噬作用內(nèi)吞CNPs,細(xì)胞內(nèi)的CNPs包裹于囊泡內(nèi),細(xì)胞線粒體腫脹,并可以觀察到自噬、細(xì)胞凋亡及細(xì)胞壞死等征象。4、CNPs干預(yù)后Bcl-2蛋白的表達(dá)下降,而Bax的表達(dá)明顯升高,自噬指示蛋白LC3-B和Beclin-1表達(dá)水平明顯增高,通路蛋白p-JNK/JNK表達(dá)水平增加(12h)。結(jié)論:1、草酸鈣腎結(jié)石患者的中段尿中可以培養(yǎng)出CNPs,中段尿的培養(yǎng)可以作為泌尿系有無(wú)感染CNPs的方法;CNPs可以通過(guò)內(nèi)吞囊泡的方式進(jìn)入HK-2細(xì)胞,CNPs的負(fù)電位可能在內(nèi)吞過(guò)程中起到作用;2、CNPs為晶體蛋白復(fù)合物,其復(fù)制增殖是一種以蛋白為載體的鈣磷沉積仿生行為;3、吞噬后的CNPs可通過(guò)作用于線粒體誘導(dǎo)細(xì)胞內(nèi)ROS的產(chǎn)生;4、CNPs誘導(dǎo)ROS的過(guò)度表達(dá)可導(dǎo)致HK-2的自噬與凋亡,嚴(yán)重時(shí)還可以引起細(xì)胞的壞死;5、CNPs在草酸鈣腎結(jié)石腎乳頭鈣斑的形成中起著重要的作用,CNPs的持續(xù)鈣磷沉積是鈣斑中磷酸鈣核心形成的基礎(chǔ);6、ROS-JNK通路在CNPs誘導(dǎo)HK-2細(xì)胞損傷中起著關(guān)鍵的調(diào)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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R692.4

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