【作者】 王佳；

【导师】 刘松财；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 热应激蛋白（HSPs）是机体在应激条件下产生的一组特异性蛋白质，能使机体迅速适应环境的变化。热应激蛋白的功能主要是作为分子伴侣，促进胞内蛋白的正确折叠，防止其聚集，并辅助未正确折叠的蛋白进入其相应的降解途径。热应激蛋白在皮肤组织伤口愈合过程中的生物学作用主要是通过对炎症反应、伤口坏死组织清理、细胞增殖和迁移及胶原蛋白合成的调节来实现的，这种作用是不可或缺的。热应激蛋白异常表达会导致创伤难以愈合，不同的热应激蛋白在创伤愈合中发挥的作用不同，而关于其在创伤愈合中的研究又缺乏系统性的比较，因此，本研究以BALB/c小鼠作为实验动物模型，将经石蜡组织切片法筛选变化差异显著（P<0.01）的3天（3d）组作为研究对象，选取已公布的84个热应激蛋白家族基因，制备成实时荧光定量PCR微阵列（qPCR Array），差异表达结果表明：Hsp90aa1、Hspa1a、Dnaja3表达上调，差异显著（P<0.05）；Bag3、Bag4表达下调，差异显著（P<0.05）。对差异表达的基因进行Gene Ontology（GO）分析表明：Hsp90aa1可能通过表达上调参与炎症反应，与HSPs其它家族因子相互作用，参与细胞调控和细胞内信号传递，并促成愈伤组织内成纤维细胞的迁移，加速伤口的愈合；Hspa1a表达上调抑制炎症反应发生，保证创伤愈合过程中新合成蛋白的正确构象；Dnaja3基因与细胞凋亡有关，且存在两种作用截然相反的不同变构体，上调意义有待进一步阐明；Bag3与Bag4基因在创伤组织愈合早期阶段的下调表达可能具有促进炎症细胞凋亡及促进创伤组织愈合的作用。信号通路检索表明：Hspa1a基因参与MAPK信号通路，Hsp90aa1参与NOD样受体信号通路，两通路可能与创伤调节有关。在蛋白表达水平上，对Hsp90aa1、Hspa1a进行蛋白印迹检测，结果表明其蛋白水平和mRNA水平的表达具有一致性。本研究表明，qPCRArray可作为高通量筛选小鼠热应激蛋白差异表达基因的一种方法，具有灵敏度高、重复性好等特点。本实验筛选到的主效基因为深入研究热应激蛋白家族因子在创伤中的作用机制提供了实验依据，其参与的生物学途径为临床上寻找促进创伤愈合的治疗方案提供了理论依据，而构建的qPCRArray也为后续系统性研究不同应激条件下热应激蛋白的差异表达开辟了新的途径。更多还原

【Abstract】 Heat shock proteins (HSPs) are a group of specific proteins, which express inresponse to various biological stresses, to preserve cell survival under adverseenvironmental conditions. HSPs function primarily as molecular chaperones,facilitating the folding of other cellular proteins, preventing protein aggregation, ortargeting improperly folded proteins to specific pathways for degradation.Bymodulating inflammation, wound debris clearance, cell proliferation, migration andcollagensynthesis, HSPs are essential for normal wound healing of the skin.The abnormal expression of HSPs may contribute to wound healing deficiencies,and there are differences in the roles played in wound healing of different HSPs.Furthermore, there is a lack of systematic comparison research on HSPs in woundhealing. In this study, BALB/c mice were used as the experimental animal model.3days (3d) group after wound was screened as the research object for inflammation,which3day was the most intense and the difference of which was significant (P<0.01)compared with the other time points.84heat shock protein family genes were selectedto prepare real-time quantitative PCR microarray (qPCR array). The results ofdifferential expression showed that Hsp90aa1, Hspa1a, Dnaja3were up-regulated andsignificant differences (P<0.05), Bag3, Bag4were down-regulated and significantdifferences (P<0.05).GO (Gene Ontology) analysis of differentially expressed genes showed thatHsp90aa1might participate in inflammatory reactions through up-regulatedexpression, interact with other HSPs family factors, and be involved in cell regulationand intracellular signaling, then promote migration of fibroblasts to accelerate woundhealing; Hspa1a up-regulation inhibits the inflammatory response to ensure the correct conformation of newly synthesized proteins in the wound healing process;Dnaja3, which has two isoforms with sharply contrasting functions, is related toapoptosis, and its up-regulating significance needs further interpretation; Thedown-regulation of Bag3and Bag4genes in the early stages of wound healing maypromote apoptosis of inflammatory cells and the healing of the wound tissue.Signaling pathway hypothesized that Hspa1a gene was involved in the MAPKsignaling pathway and Hsp90aa1gene was involved in NOD-like receptor signalingpathway, which might be related to adjustment of wound healing.Western blot analysis showed that the expression of Hsp90aa1and Hspa1a at theprotein level consisted with mRNA level.This study showed that the qPCR Array could be used to detect thehigh-throughput expression of mouse heat shock protein genes, with high sensitivityand good repeatability. The major gene of heat shock protein family factor in ourstudy can provide the experimental basis for in-depth study in trauma mechanism, andthe biological pathways that they are involved in can provide the theoretical basis forseeking treatment program to promote wound healing in clinical practice, and theqPCR Array built in this study will provide a new way to systematically study thedifferential expression of heat shock proteins in different stress conditions.更多还原