非基因毒应激对小G蛋白RhoB表达的诱导作用、机制及生物学意义

发布时间：2017-12-30 19:08

  本文关键词：非基因毒应激对小G蛋白RhoB表达的诱导作用、机制及生物学意义　出处：《第二军医大学》2008年博士论文　论文类型：学位论文

  更多相关文章： 热应激 烫伤 缺氧 小G蛋白RhoB 细胞凋亡 p38 MAPK

【摘要】： 应激是机体处于不利环境和遇到有害刺激时所产生的防御或适应性反应,应激反应发生在整体和细胞层面,后者也称为细胞应激。糖皮质激素(GC)是体内最重要的应激激素,其作用主要是由糖皮质激素受体(GR)介导的,应激时GC分泌增多对机体抵抗有害刺激起着极为重要的作用。动物实验表明,切除双侧肾上腺后,极小的有害刺激即可导致动物死亡,动物几乎不能适应任何应激环境。除了在整体应激中发挥重要作用(如调节代谢、通过允许作用调节血压等)外,已有较多体外实验显示,GC能够保护多种细胞免受应激原所致的损伤和凋亡,受GC/GR调节的蛋白在此过程中可能发挥了重要作用,如已证明一些能被细胞应激诱导、并在细胞应激中发挥重要作用的应激蛋白,如热休克蛋白70,也能被GC/GR诱导。小G蛋白RhoB是近年来我们发现的一个新的GC/GR诱导蛋白。国外近年来的研究表明,RhoB是一个早期反应基因,能够被多种基因毒应激原(致DNA损伤),如放射线、紫外线、抗癌药(如5-氟脲嘧啶、顺铂)等诱导或使其激活,并有报道RhoB上调能够促进细胞存活。但是非基因毒应激是否也影响RhoB的表达尚不十分清楚。考虑到RhoB是GR调节蛋白,因此推测该蛋白有可能也在非基因毒应激中起作用,本课题从在体和离体两个水平观察了热应激和低氧应激这两种常见的非基因毒应激对RhoB表达的影响,并进一步研究了热应激影响RhoB表达的机制以及可能的生物学意义,以期为临床烧伤、休克等疾病的救治提供新的靶点。 一、热应激对RhoB表达的诱导作用、机制及生物学意义 (一)热应激能诱导RhoB的表达 我们首先制备了30%体表面积Ⅲ度烫伤大鼠模型,采用半定量RT-PCR、Real-time PCR及Western blot方法检测了大鼠肝和肺组织中RhoB mRNA及蛋白的表达情况。结果发现,大鼠肝,肺组织中RhoB mRNA在烫伤后2h时即上调,4h时达到最大值,分别为对照组的7.5及5.7倍(p＜0.01);RhoB蛋白在烫伤后8h表达量最高。 由于烫伤后体内会产生一系列的神经内分泌反应,而我们以往的实验也证实GC可以诱导RhoB的表达。为排除体内的神经内分泌因素的影响,确定热应激本身是否可直接上调RhoB,我们进一步观察了热应激对体外培养的细胞中RhoB表达的影响。与整体实验采取肝、肺组织作为研究对象相对应,体外实验中选用了人肝癌HepG2细胞及人肺腺癌A549细胞;同时考虑肝、肺组织中除了实质细胞以外,还含有大量的组织巨噬细胞,因此我们还选取了小鼠巨噬细胞系RAW264.7作为研究对象。结果显示,热应激本身能以时间依赖性的方式快速地诱导这三种体外培养细胞中RhoBmRNA和蛋白的表达。上述实验表明,热刺激在体内和体外都可以上调RhoB的表达,且这种诱导作用无明显的组织细胞特异性。 (二)热应激诱导RhoB表达的机制研究 1.热应激上调RhoB mRNA的表达与增强其稳定性有关 我们首先采用放线菌素D(5μg/ml)抑制基因的转录,观察了热应激对RhoBmRNA稳定性的影响,以明确热应激诱导RhoB的表达是发生在转录水平,还是在转录后水平。结果显示,在A549细胞中热应激能使RhoB的半衰期由3.95h增加至7.1h(p＜0.01),在RAW264.7细胞中RhoB的半衰期则由3.98h增至7.6h(p＜0.01),表明热应激诱导RhoB mRNA的表达主要由RhoB的稳定性增加所致。 2.应激激活的p38 MAPK通路参与了热应激对RhoB的诱导作用。 已证实p38 MAPK信号通路能被包括热应激在内的多种应激原激活,我们因而观察了p38 MAPK的抑制剂SB203580对热应激诱导RhoB表达的影响。结果显示,于A549细胞的培养液中加入SB203580(10μM)抑制p38 MAPK的激活后,再将细胞进行热应激处理,RhoB的诱导水平比未经SB203580处理的细胞降低了约40%(p＜0.01);在RAW264.7细胞中RhoB的诱导水平降低了约50%(p＜0.01),表明p38 MAPK通路的激活参与了热应激对RhoB的诱导作用。 (三)热应激诱导RhoB表达的生物学意义及可能的机制 1.RhoB上调能够抑制热应激诱导的细胞凋亡 应激虽然是防御性反应,但严重的应激也可导致细胞凋亡。因此,我们观察了RhoB表达改变对A549细胞凋亡的影响,以探讨热应激时RhoB上调的生物学意义。结果显示,转染RhoB野生型质粒使RhoB过表达后,热应激引起的细胞凋亡比率由64.1%降至49.5%(p＜0.01),caspase3的活化也明显降低;而利用RNA干扰的方法减少RhoB表达后,热应激引起的细胞凋亡比率以及caspase3的活化明显增加,在RAW264.7细胞中也得到类似的结果,表明RhoB的上调能够抑制热应激诱导的细胞凋亡。 2.RhoB上调抑制热应激诱导细胞凋亡的可能机制 (1)RhoB的上调能够增强NF-κB的转录激活活性 已证明多种应激原可激活转录因子NF-κB,激活的NF-κB通过调节多种基因的转录,调节细胞的生长、分化、免疫和炎症反应,并在应激反应中发挥重要作用,如已证明NF-κB在多种细胞中能抵抗多种应激原导致的细胞凋亡。由此,我们进一步观察了RhoB对NF-κB转录激活活性的影响。结果显示,在A549细胞及RAW264.7细胞中,瞬时转染RhoB-wt质粒使RhoB过表达后既可增加NF-κB的基础转录激活活性,也可提高热应激时NF-κB的转录激活活性;而通过RNA干扰方法抑制RhoB表达后,则可明显抑制NF-κB的转录激活活性。提示,RhoB的上调可能通过增强NF-κB的转录激活活性发挥抗凋亡的作用。 (2)热应激时RhoB的上调不影响热休克蛋白70(HSPT0)的表达 HSP70是一种可由热及其他应激原诱导产生的非特异性细胞保护蛋白,可以增强机体对应激的耐受性,提高细胞生存率。大量研究表明,HSP70对射线(如γ-射线)、TNF-α等多种刺激诱导的细胞凋亡有抑制作用。因此,我们也观察了RhoB表达的改变对HSP70表达的影响。在对A549细胞和RAW264.7细胞的研究中均发现,无论是RhoB过表达还是通过RNA干扰方法使其表达受抑制,均不影响HSP70的表达,表明HSP70并未参与RhoB对热应激诱导的细胞凋亡的抑制作用。 二、低氧对RhoB表达的诱导作用 我们首先将SD大鼠放入含8%O_2、92%N_2的混合气体的低氧舱内复制急性缺氧大鼠模型,采用Real-time PCR及Western blot方法检测了大鼠脾脏和肺组织中RhoBmRNA及蛋白的表达情况。结果发现,低氧能够以时间依赖性方式诱导大鼠脾,肺组织中RhoB mRNA和蛋白的表达。继而,我们又观察了低氧对体外培养的人肺腺癌A549细胸及小鼠巨噬细胞系RAW264.7细胞中RhoB表达的影响。结果显示,低氧本身也能够以时间依赖性方式上调两种细胞中RhoB mRNA和蛋白的表达。以上结果表明,低氧这个非基因毒应激原也能够在体内和体外实验中诱导RhoB的表达,且这种诱导作用没有组织和细胞特异性。 综上所述,本实验首次发现,热应激、低氧这两个非基因毒应激原均能够在整体水平和细胞水平诱导RhoB的表达,且这种诱导没有明显的组织和细胞的特异性。热应激诱导RhoB的表达主要与其明显提高RhoB mRNA的稳定性有关,而热应激时p38 MAPK通路激活参与了RhoB表达的上调。RhoB的上调能够抑制热应激诱导的细胞凋亡,其机制并不是由HSP70介导的,而可能与RhoB增强NF-κB的转录激活活性有关。本课题所得的结果提示,RhoB具有细胞应激蛋白的特征。
[Abstract]:Stress is the body in the unfavorable environment and have encountered harmful stimuli of defense or adaptive response, stress response and cell in the overall level, the latter is also known as cell stress. Glucocorticoid (GC) is the most important body of stress hormones, which is mainly composed of glucocorticoid receptor (GR) mediated when the stress, the increase of GC secretion of the body against harmful stimuli plays a very important role. The animal experiment showed that adrenalectomy, minimal harmful stimulation can lead to the death of the animal, the animal can hardly adapt to any environmental stress. In addition to play an important role in the overall stress (such as regulating metabolism, by allowing the effect of regulating blood pressure there have been many shows, etc.) in vitro, GC can protect cells from a variety of stressors caused by injury and apoptosis may play an important role in this process by regulating protein GC/GR, such as It have been proved that some cells can be induced by stress, and play an important role in stress protein in cell stress, such as heat shock protein 70, can also be induced by GC/GR. The small G protein RhoB is a new GC/GR in recent years, we found that the induced protein. Recent studies abroad show that RhoB is an early response genes can be a variety of genotoxic stress (original induced DNA damage), such as radiation, ultraviolet radiation, anti-cancer drugs (such as 5- fluorouracil, cisplatin) or induced the activation of RhoB has been reported to increase and promote cell survival. But non genotoxic stress also affect the expression of RhoB is not very clear considering RhoB is GR regulatory protein, therefore speculated that this protein may also play a role in non genotoxic stress, the effects of heat stress and hypoxia of the two kinds of non genotoxic stress on the expression of RhoB in vivo and in vitro from two levels, We further studied the mechanism of heat stress affecting the expression of RhoB and its possible biological significance, in order to provide new targets for the treatment of clinical burn and shock.
The inducement, mechanism and biological significance of heat stress on RhoB expression
(1) heat stress can induce the expression of RhoB
We first made scald rat model of 30% body surface area were prepared by semi quantitative RT-PCR, Real-time PCR and Western blot method to detect the expression of mRNA protein and RhoB in liver and lung tissues of rats. The results showed that rat liver and lung tissue in RhoB mRNA in 2h after scald is raised to the maximum value of 4h, the control group respectively 7.5 and 5.7 times (P < 0.01); RhoB protein in rats after the expression of 8h was highest.
Because the body will produce a series of neuroendocrine responses after scald, and our previous experiments also confirmed the expression of GC can induce RhoB. Effects of neuroendocrine factors from the body, to determine whether the heat stress itself can be directly up-regulated RhoB, we further examined the effects of heat stress on the expression of RhoB in cultured cells. And the whole experiment to the liver, lung tissue as the research object corresponding to human hepatoma HepG2 cells and lung adenocarcinoma A549 cells were selected in vitro; considering the liver and lung tissues in addition to parenchymal cells, but also contains a large number of tissue macrophages, so we also selected a mouse macrophage cell line RAW264.7 as the research object. The results showed that heat stress itself in a time-dependent manner rapidly induced by these three kinds of in vitro expression of RhoBmRNA and protein in the cell. The experimental results show that the heat The expression of RhoB can be up-regulated in both in vivo and in vitro, and there is no obvious tissue cell specificity in this induction.
(two) study on the mechanism of heat stress induced RhoB expression
1. the expression of RhoB mRNA up regulated by heat stress is related to the enhancement of its stability
We used actinomycin D (5 g/ml) transcription suppressor gene, to observe the effect of heat stress on the stability of RhoBmRNA, the expression of RhoB induced by heat stress is clear at the transcription level, or at the post transcriptional level. The results showed that heat stress in A549 cells can make the half-life of RhoB by 3.95h increased to 7.1h (P < 0.01), RhoB in RAW264.7 cells the half-life increases from 3.98h to 7.6h (P < 0.01), showed that the expression of RhoB mRNA induced by heat stress mainly by the stability of RhoB increased.
2. the p38 MAPK pathway activated by stress participates in the induction of heat stress on RhoB.
It has been confirmed that p38 MAPK signaling pathway can be a variety of stress including heat stress, the original activation, we observed the effect of p38 MAPK inhibitor SB203580 induced expression of RhoB in heat stress. The results showed that SB203580 culture solution was added to A549 cells in p38 (10 M) inhibited the activation of MAPK, then the cells were heat stress, induced level of RhoB than the untreated SB203580 cells was reduced by 40% (P < 0.01); the level of RhoB induced in RAW264.7 cells was decreased by about 50% (P < 0.01), p38 showed that the activation of MAPK pathway involved in the induction of heat stress on RhoB.
(three) the biological significance and possible mechanism of heat stress induced RhoB expression
1.RhoB up regulation can inhibit the apoptosis induced by heat stress
Although the stress is a defensive reaction, but severe stress can lead to apoptosis. Therefore, we observed the changes of expression of RhoB effect on apoptosis of A549 cells, the biological significance of RhoB increase of heat stress. The results showed that the transfection of wild-type RhoB plasmid RhoB expression, apoptosis rate induced by heat stress 64.1% to 49.5% (P < 0.01), the activation of Caspase3 also decreased significantly; while using RNA interference to reduce the expression of RhoB after activation of heat stress induced apoptosis and the ratio of Caspase3 increased obviously, similar results are also obtained in RAW264.7 cells showed that the upregulation of RhoB can inhibit apoptosis induced by heat stress.
2.RhoB up-regulated the possible mechanism of inhibiting apoptosis induced by heat stress
(1) up regulation of RhoB can enhance the transcriptional activation activity of NF- kappa B
It has been proved that many stressors can activate NF- transcription factor kappa B, activation of NF- kappa B by transcriptional regulation of many genes that regulate cell growth, differentiation, immune and inflammatory responses, and play an important role in stress responses, such as the NF- kappa B has been shown in many kinds of cells can lead to apoptosis resistance to various stressors. Therefore, we further examined the effect of RhoB on activation of NF- kappa B transcription activity. The results showed that in A549 cells and RAW264.7 cells, transfected RhoB-wt plasmid that overexpression of RhoB can increase the basal transcription NF- kappa B activation activity, can also be the transcription of NF- kappa B activation activity increased during heat stress; and the inhibition of RhoB expression by RNA interference method, transcription is inhibited NF- kappa B activation activity. Suggest that upregulation of RhoB may enhance the transcription of NF- kappa B activation play anti apoptotic effect.
(2) the up-regulation of RhoB did not affect the expression of heat shock protein 70 (HSPT0) during heat stress
HSP70 is a nonspecific cell protect protein by heat and other stressors induce, can enhance tolerance to stress, improve the survival rate of cells. Many studies showed that HSP70 of radiation (such as gamma rays), TNF- alpha and other stimulation induced apoptosis is inhibited. Therefore, we also observed the effect of RhoB expression on the expression of HSP70 were found in a study of A549 cells and RAW264.7 cells, whether overexpression of RhoB or by RNA interference method to make the inhibition of the expression, did not affect the expression of HSP70 showed that the inhibitory effect of HSP70 was not involved in RhoB on apoptosis induced by heat stress.
Two, the induction of hypoxia on the expression of RhoB
We first copy the rat model of acute hypoxic hypoxia cabin SD rats in the mixed gas containing 8%O_2,92%N_2 in the detection of the expression of RhoBmRNA and protein in spleen and lung tissue in rats with Real-time PCR and Western blot method. The results showed that hypoxia can be time dependent manner to induce rat spleen, the expression of RhoB mRNA and protein in the lung tissue. Then, we also observed the effect of hypoxia on the expression of RhoB RAW264.7 cells in cultured human lung adenocarcinoma A549 cells and mouse macrophage cell line. The results showed that the expression of hypoxia itself in a time dependent manner by two kinds of cells in RhoB mRNA and protein. The above results this indicates that hypoxia non genotoxic stress is able to induce RhoB expression in vivo and in vitro, no specific tissues and cells and this effect.
To sum up, for the first time found that the experimental heat stress, hypoxia of the two non genotoxic stress were able to induce RhoB expression in the overall level and cell level, and the inducible no obvious tissue and cell specificity. The expression stability of heat stress induced by RhoB and RhoB mRNA mainly is higher, while the heat the stress of p38 MAPK activation is involved in the upregulation of.RhoB RhoB expression upregulation can inhibit apoptosis induced by heat stress, the mechanism is not mediated by HSP70 and RhoB, which may enhance transcription of NF- kappa B activation activity related to this topic. The results suggest that RhoB has the character of cell stress proteins.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R363

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