HSP70表达抑制对LPS所致细胞因子表达的影响及其机制研究

发布时间：2018-05-06 23:28

本文选题：RNA干扰 + 细胞因子　；参考：《中南大学》2009年硕士论文

【摘要】： 严重创伤、烧伤、败血症(特别是革兰氏阴性菌败血症)及各种类型的休克常导致全身炎症反应综合征(systemic inflammatoryresponse syndrome,SIRS)发生。SIRS是临床各科十分常见、平时及战时导致死亡的危重病症。虽然SIRS的发生机制迄今尚未完全阐明,但是LPS所致炎症介质的表达和细胞内信号转导通路的活化,在其发病学中的重要作用已受到广泛关注。本研究应用RNA干扰技术,抑制RAW264.7小鼠巨噬细胞的HSP70基因表达。采用RT-PCR检测发现,大肠杆菌脂多糖(LPS)可诱导转空载体的RAW264.7小鼠巨噬细胞(NEO)产生大量IL-1β和IL-6等细胞因子。而用过表达HSP70的RAW264.7细胞进行实验,发现HSP70抑制了LPS诱导的细胞因子表达。当抑制HSP70基因的表达后,再进行LPS刺激,则细胞因子表达增多,说明抑制HSP70的表达可促进LPS诱导的细胞因子表达。由于NF-κB/IκB信号转导通路的活化在LPS所致炎症反应中发挥了重要作用,我们进一步分析了HSP70表达抑制对LPS所致NF-κB信号通路活化的影响。通过Western blot等技术检测NF-κB(p65)的核移位,我们发现抑制HSP70表达后,HSP70对NF-κB(p65)核移位(LPS刺激60 min)及LPS刺激20 min所致的IκB降解的抑制作用被解除。由于IκBα的降解涉及IκBa的磷酸化,而IκBα的磷酸化是由IκB的激酶IKK以及相应的磷酸酯酶相互影响、协同作用的结果。因此,我们进一步分析了HSP70表达抑制对细胞内磷酸酯酶2A活性的影响。在不用LPS刺激和LPS刺激30min后,RNA干扰组细胞内的磷酸酯酶2A活性低于非干扰对照组细胞。说明抑制HSP70的表达可降低细胞内的磷酸酯酶2A的活性。综上所述,HSP70可能通过增高磷酸酯酶2A活性,从而抑制LPS所致IκB的磷酸化及降解,减少NF-κB的核移位,从而抑制NF-κB活化以及细胞因子的表达。
[Abstract]:Severe trauma, burn, septicemia (especially Gram-negative bacillus septicemia) and various types of shock often lead to systemic inflammatoryresponse syndromes. Sirs is very common in clinical departments. Although the pathogenesis of SIRS has not been fully elucidated, the expression of inflammatory mediators induced by LPS and the activation of intracellular signal transduction pathway have attracted much attention. In this study, RNA interference technique was used to inhibit the expression of HSP70 gene in macrophages of RAW264.7 mice. It was found by RT-PCR detection that E. coli lipopolysaccharide (LPS) could induce the production of a large number of cytokines such as IL-1 尾 and IL-6 in macrophages of RAW264.7 mice with empty vector. However, RAW264.7 cells with overexpression of HSP70 showed that HSP70 inhibited the expression of cytokines induced by LPS. When the expression of HSP70 gene was inhibited and then stimulated by LPS, the expression of cytokines increased, indicating that inhibiting the expression of HSP70 could promote the expression of cytokines induced by LPS. Since the activation of NF- 魏 B / I 魏 B signal transduction pathway plays an important role in the inflammatory response induced by LPS, we further analyzed the effect of the inhibition of HSP70 expression on the activation of NF- 魏 B signaling pathway induced by LPS. Western blot and other techniques were used to detect the nuclear translocation of NF- 魏 B p65). We found that the inhibitory effect of HSP70 on the degradation of I 魏 B induced by NF- 魏 B p65) and 20 min induced by LPS was removed after inhibiting the expression of HSP70. The degradation of I 魏 B 伪 involves the phosphorylation of I 魏 Ba, and the phosphorylation of I 魏 B 伪 is the result of the interaction of I 魏 B kinase IKK and the corresponding phosphatase. Therefore, we further analyzed the effect of inhibition of HSP70 expression on intracellular phosphatase 2A activity. The activity of phosphatase 2A in LPS interference group was lower than that in non-interference control group after 30min was not stimulated by LPS or LPS. The results showed that inhibiting the expression of HSP70 could decrease the activity of phosphatase 2A. In conclusion, HSP70 may inhibit the phosphorylation and degradation of I 魏 B induced by LPS, decrease the nuclear translocation of NF- 魏 B, and inhibit the activation of NF- 魏 B and the expression of cytokines by increasing the activity of phosphatase 2A.
【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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