热休克预处理及HSF1对LPS所致巨噬细胞迁移及黏附的影响研究

发布时间：2018-04-05 20:24

本文选题：热休克预处理　切入点：HSF1　出处：《中南大学》2009年硕士论文

【摘要】： 细胞迁移发生于胚胎形成、机体免疫、急慢性炎症及肿瘤等多种生理及病理过程中,是炎症过程中必不可少的环节之一。炎症发生时,白细胞可以通过血管壁渗出到血管外并在局部组织中聚集,这个过程称为白细胞浸润。单核/巨噬细胞通常是到达浸润部位的主要白细胞之一。白细胞浸润是一个极为复杂的连续过程,主要由白细胞的三个生物学特性决定:黏附作用、趋化作用、变形作用。其中趋化作用是白细胞参与炎症反应的重要前驱反应。热休克反应(HSR)是机体最重要的保护机制之一。热休克因子1(HSF1)通过诱导热休克蛋白(HSPs)的表达,在细胞内源性保护中发挥重要作用。近年国内外研究发现,HSF1及HSPs也参与了对小鼠胚胎成纤维细胞及人胚胎肾细胞迁移的调节过程。这为我们研究HSR及HSF1调控细胞迁移提供了重要信息。然而,HSR是否可以调控炎症中重要细胞之一的巨噬细胞的迁移?HSF1是否及如何参与该调控过程?目前尚无相关研究。为探讨热休克预处理对脂多糖(LPS)所致巨噬细胞迁移及黏附的影响,本研究首先采用腹腔注射大肠杆菌LPS的小鼠模型,通过加热使小鼠肛温升至42℃并维持15min制备热休克预处理模型,采用支气管肺泡灌洗观察热休克预处理对LPS所致小鼠肺泡白细胞渗出的影响;并采用LPS体外刺激的小鼠腹腔巨噬细胞及RAW264.7巨噬细胞株为模型,应用趋化实验、划痕愈合实验及黏附实验观察热休克预处理(42℃,1h)对LPS所致巨噬细胞迁移的影响;为探讨HSF1在热休克预处理对LPS所致巨噬细胞迁移的影响中是否发挥作用,采用HSF1基因敲除小鼠制备内毒素血症模型,应用免疫组化技术观察HSF1基因敲除小鼠肺、肝组织形态学变化及巨噬细胞浸润情况;采用HSF1敲除小鼠腹腔巨噬细胞,应用趋化实验观察HSF1敲除对LPS所致巨噬细胞迁移的影响;并采用HSF1过表达的RAW264.7巨噬细胞株,应用趋化实验观察HSF1过表达对LPS所致巨噬细胞迁移的影响。结果发现:(1).LPS(12mg/kg,2h)腹腔注射促进小鼠血清中TNF-α、IL-6、IL-10的释放;LPS(600ng/ml,2h)促进RAW264.7巨噬细胞株TNF-α的释放。(2).腹腔注射LPS(12mg/kg,48h)导致小鼠肺泡灌洗液中白细胞总数及巨噬细胞数均明显增加,热休克预处理抑制了LPS所致小鼠白细胞及巨噬细胞向肺泡的迁移;采用体外趋化实验检测LPS(600ng/ml,24h)刺激RAW264.7巨噬细胞株及小鼠腹腔巨噬细胞,发现两种细胞的迁移均明显增加,而热休克预处理能抑制LPS所致的巨噬细胞迁移;LPS处理(600ng/ml,24h)促进两种巨噬细胞向划痕部位的迁移而热休克预处理可抑制LPS对巨噬细胞迁移的促进作用;LPS处理(600ng/ml,1h)可使上述两种巨噬细胞与细胞外基质的黏附增加,而热休克预处理可抑制LPS对巨噬细胞黏附的促进作用。(3).HSF1基因敲除废除了热休克预处理对LPS所致的小鼠肺、肝组织巨噬细胞浸润及组织损伤的保护作用,也废除了热休克预处理对LPS所致小鼠腹腔巨噬细胞迁移的抑制作用。(4).HSF1基因过表达无需进行热休克预处理即能抑制LPS所致的RAW264.7巨噬细胞迁移,而热休克处理并不能进一步增强这种抑制作用。综上所述,LPS在整体小鼠和离体巨噬细胞模型均能促进巨噬细胞的迁移;热休克预处理能抑制LPS所致的小鼠肺、肝组织损伤和巨噬细胞浸润以及LPS所致离体巨噬细胞的迁移;HSF1参与了热休克预处理对LPS所致巨噬细胞迁移的抑制作用。
[Abstract]:cell migration occurs in many physiological and pathological processes , such as embryonic development , organism immunity , acute and chronic inflammation and tumor , and is one of the necessary links in the process of inflammation .

Heat shock factor ( HSR ) is one of the most important protective mechanisms of HSR , which plays an important role in the endogenous protection of human embryonic kidney cells by inducing the expression of heat shock protein ( HSPs ) . In recent years , it has been found that HSFs and HSPs also participate in the regulation of the migration of mouse embryonic fibroblasts and human embryonic kidney cells .

In order to investigate the effects of heat shock pretreatment on the migration and adhesion of LPS - induced macrophages in mice , the effects of heat shock pretreatment on LPS - induced macrophage migration were studied by means of heat shock pretreatment .

The results showed that : ( 1 ) LPS ( 12 mg / kg , 2h ) could promote the release of TNF - 伪 , IL - 6 and IL - 10 in serum of mice . LPS ( 600ng / ml , 2h ) promoted the release of TNF - 伪 in RAW264.7 macrophage . LPS ( 600ng / ml , 24h ) stimulated the migration of macrophages and macrophages . LPS treatment ( 600ng / ml , 24h ) promoted the migration of two kinds of macrophages to scratch sites . LPS treatment ( 600ng / ml , 1h ) promoted the adhesion of two kinds of macrophages to the extracellular matrix . The effect of heat shock pretreatment on LPS - induced infiltration and tissue injury of mouse lung and liver tissue was abolished by HSFI , and the inhibitory effect of heat shock pretreatment on peritoneal macrophage migration in mice induced by LPS was also abolished . It is not necessary to perform heat shock pretreatment to inhibit the migration of RAW264.7 macrophages due to LPS , but the heat shock treatment can not further enhance the inhibition .

In conclusion , LPS can promote the migration of macrophages in both the whole mouse and the ex vivo macrophage model . Heat shock pretreatment can inhibit the migration of LPS - induced lung , liver tissue injury and macrophage infiltration and LPS - induced dissociation of macrophages .

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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