【作者】 周建辉；

【导师】 陈香美；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2010， 博士

【摘要】 背景与目的：横纹肌溶解可以导致急性肾损伤,肌红蛋白是致病的关键因素。但是,是高铁肌红蛋白还是亚铁肌红蛋白导致肾小管上皮细胞损伤,研究结果并不一致,横纹肌溶解肾损伤的确切机制也尚未阐明。在急性肾损伤过程中,常有细胞凋亡,近年研究发现内质网应激在细胞凋亡中具有重要作用。肌红蛋白可以引起肾小管上皮细胞凋亡,但此过程是否由内质网应激所诱导,以及相关的凋亡信号传导通路,尚未明确。本研究通过对比分析高铁和亚铁肌红蛋白对HK-2细胞的作用,证实配位铁价态对肌红蛋白细胞毒性的影响。进而以亚铁肌红蛋白诱导HK-2细胞凋亡,检测内质网应激及相关凋亡蛋白的表达,解析肌红蛋白诱导凋亡的内质网应激机制。最后,以抗体为配基构建肌红蛋白免疫吸附实验治疗装置,初步验证其清除功能。方法：1.分析肌红蛋白配位铁价态对其肾小管上皮细胞毒性的影响：①HK-2细胞分别与高铁和亚铁两种价态的肌红蛋白孵育,以MTT试验评估细胞活性,细胞培养上清乳酸脱氢酶(LDH)检测评价细胞损伤程度,分析肌红蛋白配位铁价态对其细胞毒性的影响。②不同剂量亚铁肌红蛋白孵育HK-2细胞,以MTT试验评估细胞活性,培养上清LDH检测评价细胞损伤,Hoechst染色评估细胞凋亡,分析亚铁肌红蛋白肾小管上皮细胞毒性的剂量效应关系。2.探索肌红蛋白诱导肾小管上皮细胞凋亡的内质网应激机制：①亚铁肌红蛋白孵育HK-2细胞,Western blot检测孵育后各时间点的内质网应激和相关凋亡蛋白的表达,分析可能参与凋亡的信号途径。②在阻断内质网IP3R钙通道的基础上,以亚铁肌红蛋白孵育HK-2细胞,Western blot检测内质网应激和相关凋亡蛋白表达的变化,流式细胞仪检测细胞凋亡率,分析肌红蛋白诱导HK-2细胞凋亡的内质网应激机制。3.构建肌红蛋白免疫吸附实验治疗装置：①利用生物分析软件筛选并合成肌红蛋白抗原肽段,经免疫、纯化获得肌红蛋白多克隆抗体。②经纯度、效价、特异性检测,选取优势肽段与筛选的载体交联,制备免疫吸附填料并装配免疫吸附柱。③初步验证体结果：1.亚铁肌红蛋白诱导肾小管上皮细胞损伤：①200uM亚铁肌红蛋白孵育HK-2细胞,细胞活性在48h显著降低,LDH释放量在24h显著增加；高铁肌红蛋白对HK-2细胞无显著影响。②分别以12.5-200uM亚铁肌红蛋白孵育HK-2细胞,结果显示亚铁肌红蛋白剂量与HK-2细胞活性呈负相关,与培养上清LDH释放量呈正相关,与细胞凋亡率呈正相关。2.肌红蛋白通过内质网应激介导肾小管上皮细胞凋亡：①200uM亚铁肌红蛋白孵育HK-2细胞,内质网伴侣蛋白Grp78在3-12h维持较高表达,线粒体凋亡途径启动者caspase-9在12-24h维持较高表达,而内质网特异性凋亡途径启动者caspase-4表达量与对照组无显著差异。②阻断内质网IP3R钙通道同时给予亚铁肌红蛋白孵育,与单用亚铁肌红蛋白孵育相比较,Grp78表达进一步增高,caspase-9显著抑制,caspase-4显著增高,HK-2细胞凋亡率无显著改变。3.构建肌红蛋白免疫吸附实验治疗装置：①设计、合成2条抗原肽段L-12与D-12,偶联大分子蛋白KLH并免疫大耳白兔,抗血清经纯化获得2种兔抗大鼠肌红蛋白多克隆抗体,抗L-12和抗D-12。②SDS-PAGE电泳检测纯度,ELISA检测效价,Western blot和免疫组化检测抗体特异性,选取抗D-12抗体与筛选的D-380树脂交联,制备免疫吸附填料并装配1ml和5m1吸附柱。③泵管系统模拟体外循环验证结果显示吸附柱有较好的清除功能并可以复用。快速蛋白液相色谱仪验证结果显示吸附柱对肌红蛋白的吸附率显著高于其它蛋白(白蛋白和溶菌酶)。结论：1.配位铁价态决定了肌红蛋白的细胞毒性,是亚铁肌红蛋白而非高铁肌红蛋白引起肾小管上皮细胞损伤,其抑制细胞活性、产生细胞损伤、诱导细胞凋亡的作用呈剂量依赖性。2.亚铁肌红蛋白诱导肾小管上皮细胞发生内质网应激,进而通过依赖caspase-9的线粒体途径介导细胞凋亡。内质网IP3R钙通道阻断时,依赖caspase-4的内质网特异性途径可能成为介导细胞凋亡的替代途径,提示单纯阻断线粒体途径不能抑制凋亡。3.以肌红蛋白抗体为配基构建的免疫吸附实验治疗装置对肌红蛋白具有优良的清除功能。更多还原

【Abstract】 Backgroundst Objective:About 13-50% of rhabdomyolysis is complicated with AKI and myoglobin is the key element. Literatures about myoglobin toxicity mainly focus on oxidative stress, but the results diverse. The excact mechanisms of myoglobin induced kidney injury remains unclear. Apoptosis often occurs in animal models of AKI or cultured cells. Endoplasmic reticulum stress (ERS) has recently been found to play an important role in apoptosis. Myoglobin is able to induce apoptosis in tubular epithelial cells, but whether it is mediated by ERS and the signaling pathways are still unknown. In this study we mainly focus on myoglobin renal toxicity and the ERS mechanisms in myoglobin induced tubular epithelial cell apoptosis, then construct experimental immunoadsorbent devices.Methods:1.Valent states of coordinated iron affect myoglobin toxicity:i)HK-2 cells were incubated with ferrous or ferric myoglobin, respectively, then cell viability was evaluated by MTT assay, cell injury by LDH release test. ii)HK-2 cells were incubated with different doses of ferrous myoglobin to analyse the dose-effect relationships.2.ERS mechanisms in myoglobin induced apoptosis: i)HK-2 cells were incubated with ferrous myoglobin and proteins relating to ERS or apoptosis were analysed by western blot. ii)With IP3R calcium channel blocked by 2-APB, HK-2 cells were incubated with ferrous myoglobin. Then we determined protein expression of Grp78, caspase-4, caspase-9, caspase-3 by western blot and evaluated cell apoptosis by flow cytometry.3.Construct inmmunoadsorbent devices:i)Achieved the purified rabbit anti-rat myoglobin polyclonal antibodies. ii)Prepared immunoadsorbent columns. iii)Verified the adsorption capacity in vitro.Results:1.Ferrous myoglobin cell toxicity:i)HK-2 cells incubated with ferrous myoglobin showed lower viability, more cell injury while ferric myoglobin didn’t affect HK-2 cells. ii)Ferrous myoglobin dosage was correlated positively with cell LDH release and apoptosis rate, and inversely with cell viability.2.ERS mediated tubular epithelial cell apoptosis:i)Incubated with 200uM ferrous myoglobin, Grp78 increased and remained at high level in 3-12h while caspase-9 remained high in 12-24h. Caspase-4 was non significant to control. ii)Compared to merely myoglobin incubation,2-APB plus myoglobin incubation showed further more Grp78 expression, inhibited caspase-9, dramatically increased caspase-4, and cell apoptosis wasn’t ameliorated.3.Immunoadsorbent devices:i)Synthesized 2 antigen peptides, L-12 and D-12. Linked with KLH and immuned rabbits, obtained 2 polyclonal antibodies, anti-L-12 and anti-D-12 pAb. ii)Determined purity, titer and specificity of the antibodies, then corsslinked anti-D-12 pAb with D-380 resin to prepare adsorbent fillings. Then assemble immunoadsorbent devices. iii)The mimic extracorporeal system verification indicated that the immnoadsorbent column had good function and reusable. FPLC verification showed the removal of myoglobin was significantly more than that of other proteins, i.e., BSA or lysozyme.Conclusions:1.Valent state of cocordinated iron in myoglobin determines its cell toxicity. It is ferrous myoglobin that causes tubular cell injuries and the toxicity is dose-dependent.2.Ferrous myoglobin can induce ERS, then mediates cell apoptosis by caspase-9 dependent mitochondrial pathway. When IP3R calcium channel is blocked, caspase-4 dependent endoplasmic reticulum specific pathway may serve as a substitutive apoptosis pathway.3.With myoglobin polyclonal antibody as the ligand, the immnoadsorbent devices have good adsorption capacity.更多还原