【作者】 张志清；

【导师】 钱令嘉；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2009， 博士

【摘要】 适应(adaptation)是生物体应对生存环境调整自身生理机制以适应环境而生存的过程,是对抗应激损伤的有效机制。应激状态下,机体通过神经内分泌系统的调节,多层次启动了复杂的细胞内调控网络,导致多种内源性物质产生,如腺苷、受体、细胞内信息传导因素、效应蛋白等,以维持机体内环境的相对稳定,修复分子损伤,减轻细胞损伤的发生,增强机体对外界不良因素的抗御能力,逐步建立了对外界应激源的适应能力。热休克蛋白(heat shock/stress proteins, HSPs)是机体应激时迅速大量表达的一组蛋白质,HSP70是HSP家族中最著名的成员,作为分子伴侣参与了新生肽链的折叠、转运和蛋白质的组装聚集及降解。更值得关注的是,HSP70可以通过阻抑细胞内重要蛋白质的变性和聚集,修复已变性失活的蛋白质,稳定细胞骨架、增强细胞内抗氧化能力、保护酶功能以抗御细胞损伤,是重要的损伤修复分子。本研究室前期工作发现,用预热应激方法诱导HSP70高表达能够明显减缓应激损伤,加速机体应激适应机制的建立;进一步的研究发现,HSP70的升高显著阻抑应激所致细胞凋亡途径。因此我们推测,HSP70是应激适应建立的重要功能蛋白质,HSP70与其所调节的多种功能蛋白质及其所参与的细胞分子修复机制构成了一种多系统多层次协调的应激损伤修复体系,为应激适应机制的建立提供了可能性和生物学基础。本研究通过认识HSP70在机体应激适应过程中表达规律的变化,确认其对应激适应的促进功能,发现HSP70在此过程中的相互作用蛋白质,阐明其生物学功能及其相互关系,不仅可以深刻揭示应激适应的生物学机制及其重要蛋白质基础,而且对于探索控制应激损伤,促进应激适应的医学措施具有十分重要的意义。一、HSP70在应激适应建立过程中的表达规律及其生物学功能采用间歇性温和束缚应激的方式诱导大鼠应激适应机制的建立,以机体在应激状态下神经内分泌变化和心肌的损伤程度作为确认大鼠应激适应机制建立的重要指标。结果表明,适应大鼠应激时,HPA轴反应趋于平缓,血浆糖皮质激素、儿茶酚胺分泌水平、LDH、CK-MB活性显著低于单纯应激组;Nagar olsen染色和电镜检查可见:适应大鼠应激时心肌损伤明显低于单纯应激组,表明应激适应动物模型已经建立。同时,模拟适应机体糖皮质激素水平变化,采用低浓度糖皮质激素预处理心肌细胞株,发现其可以降低高浓度糖皮质激素对心肌细胞的损伤作用,表明低浓度糖皮质激素预处理可以诱导心肌细胞应激适应的建立。采用蛋白质免疫印迹技术观察应激适应大鼠心肌组织中HSP70表达水平。结果显示,间歇性温和应激诱导心肌组织HSP70表达水平显著升高,并在适应诱导过程中持续高水平表达,在HSP70表达水平升高的应激适应状态下,适应大鼠应激时心肌损伤明显低于单纯应激组。离体细胞研究发现,低浓度糖皮质激素预处理亦可导致心肌细胞HSP70明显升高。调控心肌细胞中HSP70的表达,可见高表达HSP70对糖皮质激素导致的心肌细胞损伤具有明显的抑制作用,而抑制HSP70表达可显著增强糖皮质激素导致的心肌细胞损伤。表明HSP70在应激适应建立中具有重要的生物学功能。二、应激适应建立过程中HSP70相互作用蛋白质的筛选采用生物信息学分析发现了43个与HSP70具有相互作用的蛋白质,包括HSP70协同伴侣分子:HSF1、HSPBP1、BAG1、BAG2、BAG3、BAG4、HIP、HSP40、CHIP、HOP、DNAJA3、DNAJB11、TTC1;凋亡通路相关蛋白质:PDCD8、FANCC、IKBKG、TP53、APAF1、BCL2、HCFC1、RHOA等;细胞周期调节相关蛋白质:APEX1、MAP3K5、PPP1R15A、CDK9、EIF2AK1、NR3C1、EIF2AK2、APOB、PKC等;细胞分化调控蛋白质:MSR1、PTMA、TIF1、KRT7等;胚胎发育相关蛋白质:SOX9、PEX5等;蛋白质的翻译转运相关蛋白质:BAT3、TCERG1、TID1、TOM7等;以及能量代谢调节蛋白质:NQO1、SLC5A1等。采用酵母双杂交方法,以HSP70为诱饵蛋白筛选与HSP70具有相互作用的蛋白质,发现HIP与HSP70存在相互作用。免疫共沉淀结果不仅确认应激适应过程中HIP和HSP70相互作用的存在,而且显示随着应激适应诱导时程的延长及应激适应机制的建立,HIP与HSP70相互作用复合物逐渐增多,提示了HIP和HSP70的相互作用在应激适应建立过程中可能具有重要作用。三、HSP70相互作用蛋白质HIP在应激适应建立过程中的生物学功能采用蛋白质免疫印迹技术观察应激适应大鼠心肌组织HIP的表达变化,结果显示:应激适应可诱导HIP表达水平显著升高,且在适应诱导过程中持续高水平表达。以心肌细胞株H9C2建立细胞应激适应模型,HIP在应激适应诱导建立过程中持续升高。调控心肌细胞株中HIP表达水平,可见高表达HIP对糖皮质激素导致的心肌细胞损伤具有明显的抑制作用,而抑制HIP表达可导致心肌细胞损伤明显增强;表明HIP在应激适应建立中亦具有重要的生物学功能。对HIP和HSP70在H9C2细胞中表达水平实现共同调控,观察其对应激细胞的保护作用。实验结果显示,应激适应建立过程中HIP生物学功能的实现依赖于HSP70的存在;抑制HSP70表达,或使HIP与HSP70相互作用结构域缺失不能与HSP70结合,则使HIP失去对应激心肌细胞的保护作用。应用荧光素酶报告基因系统的初步研究结果显示,HIP与HSP70阻抑应激心肌细胞损伤可能是通过降低糖皮质激素的转录激活活性实现的。综上所述,HSP70在应激适应建立过程中具有重要的生物学功能;HIP是应激适应过程中心肌细胞内与HSP70相互作用的重要蛋白质,其可通过HSP70的介导,降低糖皮质激素受体的转录激活活性,实现对应激机体心肌细胞的保护作用,促进应激适应机制的建立。更多还原

【Abstract】 Adaptation is characterized as stressful elicit a complex array of behavioral and physiological changes believed to contribute to optimal coping of the organism with the situation. As a result of stress, cellular and molecular response could result in either adaptation or damage in cells, which depended on the stress load. This implied that biomolecule that can inhibit and repair stress damage were just the important regulating molecule and proteins. HSPs(heat shock/stress protein, HSP)was a group of proteins which were rapidly expressed with organism coping with stress, including HSP90、HSP70、HSP40 and small HSP. HSP70 was a very famous member among HSP family, it played important roles in nascent polypeptide chain folding and transport and protein assembly and degradation, and HSP70 could also regulate biological function of many proteins and vital process, especially normal cell signaling and cell life cycle regulation. It must be paid much more attention that HSP70 could inhibit important cellular protein degeneration and degradation, repair damaged protein, stabilize cytoskeleton, improve cellular anti-oxidant ability, and protect enzyme activity, so HSP70 was a very important restoration protein. Previous study of our lab had demonstrated that high expressing level of HSP70 with heat shock can inhibit cell apoptosis to improve adaptation establish. So we presumed that HSP70 was an important protein which composed a system of protein damage and restoration with its regulation proteins to improve adaptation establish. So to explore the HSP70 expression pattern during adaptation, confirm its adaptation promotion, detect HSP70 interacting protein during adaptation, and interpret its biological function could not only deeply reveal biological mechanism of adaptation and its protein basis, but also have important significance in controlling stress damage and promoting adaptation.1. Expression pattern of HSP70 during adaptation and protecting role of HSP70 during stress damage. Intermittence chronic restraint stress was used to induce the construction of adaptation, and neuroendocrine hormone and myocardium damage degree were used as indexes to evaluate adaptation establish. The results showed that HPA response tend to be mild when adaptation rat were given a more intense stress. And plasma level of E、NE、GC and plasma LDH\CK-MB activity of stress group after adaptation were lower than more intense stress group. pathological observation showed that adaptation could significantly attenuate the damage of more intense stress. So those results showed that an adaptation model was established. At the same time, low GC concentration pretreatment can attenuate the damage of high GC concentration treatment, so an adaptation cell model was established.HSP70 level was detected by western blotting. The results showed that HSP70 level continuing increased within two weeks of construction of adaptation, adaptation rat with high HSP70 level could attenuate the damage of more intense stress. HSP70 expression pattern was similar in H9C2 cell with glucocorticoids treatment. These results implied that HSP70 have an important role in adaptation. We also found that HSP70 could protect H9C2 cell from GC inducing cell damage with over expression and inhibition of HSP70. These results confirmed that HSP70 had an important role on adaptation attenuating the damage of more intense stress.2. Screening for HSP70 interacting proteins during adaptation43 proteins were acquired with bioinformatics technology including: co-chaperones: HSF1、HSPBP1、BAG1、BAG2、BAG3、BAG4、HIP、HSP40、CHIP、HOP、DNAJA3、DNAJB11、TTC1, proteins in apoptosis pathway: PDCD8、FANCC、IKBKG、TP53、APAF1、BCL2、HCFC1、RHOA, cell cycle regulating proteins: APEX1、MAP3K5、PPP1R15A、CDK9、EIF2AK1、NR3C1、EIF2AK2、APOB、PKC, cell differentiation correlated proteins:MSR1、PTMA、TIF1、KRT7, embryo development correlated proteins: SOX9、PEX5, protein translation and transport correlated proteins: BAT3、TCERG1、TID1、TOM7, energy metabolism correlated proteins: NQO1、SLC5A1.pGBKT7-Hsp70 was used as a bait to screen cDNA library with yeast two hybrid technology. HIP was acquired after yeast two hybrid screen, repeat identification and sequencing. Co-immunonoprecipitation of complexes captured using antibody against HSP70 showed that HIP bound to HSP70 in rat adaptation animal model and H9C2 cell with GC treatment. So these results showed that HIP also had important role in adaptation with its interaction with HSP70.3. Mechanism of protection role of HSP70 and its interacting protein during stress damageWestern blotting results showed that HIP level continuing increased within two weeks of construction of adaptation. HIP expression pattern was similar in H9C2 cell with glucocorticoids treatment. HIP could also protect H9C2 cell from GC inducing damage with over expressing and inhibiting HIP expression. Mutant analysis and cotransfection research showed that HIP and HSP70 could protect H9C2 cell from GC inducing damage through their interaction. Luciferase report gene system showed that HIP and HSP70 could decrease the GR transcription activity, this may be a mechanism of HSP70 and HIP protecting H9C2 cell from GC inducing damage.In conclusion, HSP70 had important roles in adaptation establish, and HIP was an important HSP70 interacting protein in adaptation, which can reduce the transcription activity of GR, protect H9C2 cells from GC induced damage, and promote adaptation establish.更多还原