【作者】 贺全勇；

【导师】 罗成群；

【作者基本信息】 中南大学 ， 烧伤外科， 2009， 博士

【摘要】 最近研究发现,Wnt5a是一个高度特异的自分泌及旁分泌的巨噬细胞来源的效应分子,参与了炎症反应过程。Wnt5a释放到细胞外,成为一种重要的细胞因子,介导局部或全身炎症反应,成为目前感染及危重病防治研究的又一个重要分子靶点。热休克蛋白是热休克反应诱导产生的一类蛋白质,其中的诱导型热休克蛋白70(HSP70)是最重要的成员,也是诱导产生最多的成员。本研究先观察Wnt5a在烧伤感染病人血清中含量的改变,并探讨诱导型HSP70对Wnt5a表达、释放及促炎功能的影响。首先,本研究观察了Wnt5a在烧伤病人血清中的改变。结果如下:(1)烧伤病人血清中Wnt5a的含量显著高于正常对照组。动态观察发现,病情好转血清中Wnt5a含量降低,而病情恶化血清中Wnt5a持续升高;(2)烧伤病人血清中HSP70含量高于正常对照组,但病人的严重程度与血清中HSP70含量的变化关系不大。以上结果表明,Wnt5a,参与了烧伤病人炎症的发病过程,且检测Wnt5a可作为炎症严重程度的诊断指标。炎症因子主要是由微生物或微生物组份刺激单核巨噬细胞产生的,其中LPS诱导巨噬细胞产生与释放炎症因子是一个广泛采用的模型。本研究随后探讨了内毒素(LPS)及促炎因子TNF-α对Wnt5a表达释放的影响。研究发现,LPS能诱导巨噬细胞RAW264.7中Wnt5a表达上调及向胞外释放,同时能诱导促炎因子TNF-α的释放;用Wnt5a刺激巨噬细胞,能诱导TNF-α释放的显著提高;用TNF-α刺激巨噬细胞同样能诱导Wnt5a的表达释放上调。这与文献报道一致,促炎因子能促进Wnt5a的表达释放,Wnt5a又能促进其他炎症因子的表达释放。因此,本研究进一步证明Wnt5a是一个新的炎症因子,可能参与了烧伤病人炎症的发生发展过程。文献报道,抑炎因子白细胞介素(IL)-10与活化蛋白C(APC)能抑制Wnt5a的表达而抑制炎症反应,因此认为Wnt5a可以作为炎症治疗的新靶点。热休克反应(HSR)和HSP70在炎症中的作用也有一些研究。有人认为HSP70能抑制某些炎症因子的释放和表达,也有人认为,HSP70可能具有促炎功能。作为一个新发现的炎症介质,Wnt5a在炎症中功能还知道的很少。HSR和HSP70对Wnt5a的影响也都不清楚。热休克蛋白(heat shock proteins,HSPs)是一高度保守的应激蛋白家族,广泛存在于原核细胞和真核细胞中。HSP70是热休克蛋白家族中最重要的成员,其基本功能是参与新生蛋白质的折叠、解聚、移位等,故有“分子伴侣”(molecular chaperone)之称。长期以来,热休克蛋白被认为是细胞内的蛋白质,只能在细胞内发挥功能。为了观察细胞内的HSP70对Wnt5a的表达与释放的影响,在证明Wnt5a能为LPS诱导表达与释放,且Wnt5a又能刺激巨噬细胞释放其它炎症因子的基础上,本研究首次观察了HSP70对Wnt5a表达与释放及其促炎功能的影响。本研究首先构建了HSP70的表达载体。将HSP70的真核表达质粒导入巨噬细胞得到了高效表达。利用热休克预处理与转基因技术,分别探讨高表达HSP70的细胞受LPS或其他炎症因子刺激时释放Wnt5a的改变。结果发现,与未处理的细胞相比,温和的热休克(42℃)预处理后的细胞,在受到LPS与TNF-α刺激时候,(1)细胞内表达的Wnt5a降低;(2)向细胞外释放的Wnt5a减少。受LPS刺激后,转HSP70基因的细胞与转空载体的细胞相比,(1)细胞内表达的Wnt5a下降;(2)细胞向胞外分泌的Wnt5a减少。当用TNF-α代替LPS刺激细胞重复上述实验,得到类似结果。本研究同样观察了细胞内HSP70的上升对Wnt5a促炎功能的影响,发现不管是温和热休克(42℃)还是转HSP70基因,均能显著抑制Wnt5a诱导的TNF-α释放。上述结果表明,巨噬细胞内HSP70的表达上调既能显著抑制炎性刺激导致的Wnt5a的表达与释放,又能显著抑制Wnt5a的促炎功能。近年研究发现,HSP70能被释放到细胞外环境中,称为细胞外HSP70(extracellular HSP70),而且细胞外HSP70可能作为免疫系统的“危险信号”,调节免疫细胞的功能,参与免疫性疾病的病理过程。近年来研究发现,脓毒症病人血中HSP70水平明显升高。为了进一步探讨细胞外(包括血清中)HSP70对Wnt5a的影响,本研究采用重组人HSP70刺激巨噬细胞,然后观察其对Wnt5a释放的影响。结果显示,HSP70本身对Wnt5a的释放没有显著影响,但能显著抑制LPS诱导的巨噬细胞Wnt5a的释放。本研究同时检测了巨噬细胞IL-10的释放,表明细胞外HSP70能显著促进IL-10向细胞外释放。随后本研究观察了TLR2与TLR4在巨噬细胞抗炎因子IL-10分泌中的作用,发现IL-10的分泌依赖于TLR4。综上所述,烧伤患者血清中Wnt5a及HSP70水平均明显升高,并且血清中Wnt5a含量的高低与烧伤脓毒症的严重程度有关;LPS与TNF-α能促进巨噬细胞Wnt5a的表达上调与分泌上升,Wnt5a反过来又能刺激巨噬细胞释放TNF-α;热休克及转基因均能显著上调巨噬细胞内HSP70的表达,细胞内HSP70表达的上升既能抑制LPS及TNF-α诱导的Wnt5a的表达与释放,又能抑制Wnt5a诱导的TNF-α的释放;采用重组纯化的HSP70处理巨噬细胞同样能抑制LPS诱导的Wnt5a的释放,同时通过TLR4促进抑炎细胞因子IL-10的释放。因此,HSP70可能成为防治脓毒症的潜在药物。更多还原

【Abstract】 Recent study found that Wnt5a is a highly specific autocrine and paracrine macrophaged-derived effector molecule which triggers inflammation. Wnt5a secreted outside the cells becomes an important cytokine that mediates partial or full inflammation, so it has been a novel molecular target for the prevention and treatment of inflammation-related diseases. Heat shock proteins(HSPs) are a kind of proteins that are induced by heat shock response(HSR), of which heat shock protein 70 is the most important member and the most induced one. In this study the change of Wnt5a in level in burn patients are first investigated, and then the effects of HSP70 on expression and release and proinflammatory function of Wnt5a are also studied for the first time.Firstly, the changes of Wnt5a levels in sera of burn patients with sepsis were investigated. The results were: (1) the levels of serum Wnt5a of burn patients with sepsis are found to be greater than those of controls; dynamic observation showed that the serum Wnt5a concentrations were associated with the severity of burn patients, and Wnt5a levels going up or down with the severity of the burn patients. (2) the similar results were obtained when serum HSP70 levels were investigated. These results suggest that Wnt5a may take part in the process of sepsis in burn patients with sepsis and may be as a diagnostic parameter to severity of sepsis.Inflammatory cytokines are produced by monocytes/macrophages stimulated with mycobacteria or conserved bacterial structures. The use of LPS to induce macrophages to produce inflammatory cytokines is a widely applied model. In this study the investigation of the effects of LPS and TNF-αon the expression and release of Wnt5a was then performed. It was found that LPS could induce the expression and release of wnt5a from macrophage Raw264.7, and at the same time the release of the proinflammatory cytokine TNF-α; the release of TNF-αfrom raw264.7 was also induced by wnt5a, and vise versa. These results are consistent with the reports from other literatures. Therefore we demonstrated that wnt5a is a new inflammatory mediator, functioning in the processes of sepsis of burn patients. Previous study reported that the anti-inflammatory cytokine interleukin (IL)-10 and activated protein C (APC) can inhibit the expression and release of wnt5a, suggesting that wnt5a may be a new target molecule for treatment and prevention of sepsis. HSR and HSP70 were studied in the processes of sepsis. Some researchers think they inhibit the expression and release of inflammatory cytokines while others think they promote their release. As a new inflammatory mediator little is known about wnt5a in the processes of sepsis. The effects of HSR and HSP70 on wnt5a are unknown.HSPs are a superfamily of highly conserved proteins distributing throughout prokaryotic cells and eukaryotic cells, of which HSP70 is the most important member. Their basic functions are to take part in folding, disaggregation and translocalization of neonate proteins, so they are called molecular chaperone. HSPs are thought to be intracellular proteins that function only within cells during the long-term period. In order to investigate the effects of intracellular HSP70 on the expression and release of wnt5a, and based upon the facts that wnt5a can be expressed and released from macrophages by induction of LPS and TNF-α, and that wnt5a can induce the release of other cytokines such as TNF-α, this study for the first time investigated the effects of intracellular HSP70 on the expression, release and pro-inflammation of wnt5a. We first constructed HSP70 expression vector, then the expression vector was transduced into macrophage Raw264.7 for overexpression. Using HS pretreatment and gene transffection it was found: (1) overexpression of hsp70 can inhibit the expression and release of wnt5a induced by LPS; (2) when the experiment was done using TNF-αin stead of LPS the similar results were obtained; (3) the overexpression of hsp70 can inhibit pro-inflammatory function of wnt5a by inhibiting the release of other pro-inflammatory cytokines including TNF-αby induction of wnt5a. All these results demonstrated that intracellular hsp70 can significantly inhibit the expression and release of wnt5a induced by LPS and TNF-α, and also inhibit the pro-inflammatory function of wnt5a.Recent study shows that hsp70 can be released to external milieu that was called extracellular hsp70,and extracellular hsp70 may regulate the function of immune cells as a "dangerous signal" in immune systems, participating in the processes of inflammatory diseases. In recent years it was found that the levels of serum hsp70 in patients with sepsis or septic shock were higher than those in normal controls. In this study recombinant human hsp70 was used to stimulate macrophage raw264.7 so as to investigate the effects of extracellular hsp70 on wnt5a.The results showed that the recombinant purified hsp70 itself did not affect the release of wnt5a, but it can markedly inhibit the release of wnt5a induced by LPS. The anti-inflammatory cytokine IL-10 was detected at the same time, and it was found that the pretreatment of the recombinant hsp70 can promote the release of IL-10 from macrophage raw264.7. The receptors TLR2 and TLR4 were studied with the corresponding antibodies, and it was found that the recombinant hsp70 promoted the release of IL-10 in a TLR4 dependent manner.Taken together, the levels of serum wnt5a and hsp70 in burn patients significantly increased, and the concentrations of serum wnt5a may be related to the severity of sepsis of the burn patients. LPS and TNF-αincreased the production of wnt5a, and wnt5a in return stimulated the release of the proinflammatory cytokine TNF-α. HS and gene transfection can lead to the increasing expression of HSP70 in macrophages, and the intracellular hsp70 can inhibit both the expression and release of wnt5a induced by LPS and TNF-α, and the release of TNF-αinduced by wnt5a. Recombinant hsp70 can also inhibit the release of Wnt5a induced by LPS possibly by promoting the release of anti-inflammatory cytokine IL-10 in a TLR4 dependent manner.更多还原