【摘要】：研究背景隨著經(jīng)濟(jì)、社會(huì)的發(fā)展,創(chuàng)傷性腦損傷(traumatic brain injury,TBI)成為生活中致死率和致殘率較高的疾病之一[1,2]。其對患者的身心健康造成了一定程度的不利影響,也給社會(huì)帶來了嚴(yán)重負(fù)擔(dān)。TBI后腦組織代謝加速而此時(shí)受傷腦組織中的線粒體的形態(tài)、功能發(fā)生改變致其無法進(jìn)行正常的能量代謝從而使細(xì)胞的正常生理功能發(fā)生故障,最終導(dǎo)致后期更為嚴(yán)重的繼發(fā)性損傷[3]。而在此過程中,大量線粒體形態(tài)和結(jié)構(gòu)遭到破壞,導(dǎo)致線粒體無法正常供應(yīng)能量,引起神經(jīng)元細(xì)胞的死亡。所以,增強(qiáng)線粒體自身的生物合成以改善線粒體的能量代謝,可大大減少細(xì)胞的損傷,起到保護(hù)神經(jīng)元的作用。第一部分槲皮素可在TBi后激活Nrf2通路并保護(hù)線粒體損傷目的探討槲皮素通過Nrf2途徑對TBI后線粒體損傷的保護(hù)作用。方法選取雄性成年ICR小鼠,采用自由落體模型,分為四組:假手術(shù)組(sham組)、外傷組(TBI組)、外傷+溶劑組(TBI+vehicle組)、外傷+槲皮素組(TBI+quercetin組)。在傷后24h取傷灶周圍大腦皮層組織,使用Western blot檢測細(xì)胞核及胞漿的中Nrf2蛋白的表達(dá)情況;檢測線粒體及胞漿中Bax及CytC的濃度。使用免疫組化檢測Nrf2蛋白由胞漿向胞核轉(zhuǎn)移情況;用酶標(biāo)儀檢測線粒體SOD及MDA濃度。結(jié)果與假手術(shù)組相比,外傷組和溶劑組Nrf2表達(dá)增強(qiáng)且向核內(nèi)轉(zhuǎn)移明顯增多。線粒體Cyt C及SOD含量減少,而Bax與MDA含量增多。槲皮素干預(yù)后不但促進(jìn)了 Nrf2的入核和表達(dá),而且使創(chuàng)傷性腦損傷后線粒體的氧化應(yīng)激相關(guān)蛋白表達(dá)減少。。結(jié)論槲皮素可進(jìn)一步激活TBI后Nrf2信號通路,減輕TBI后線粒體的破壞。第二部分Nrf2通路在槲皮素減輕TBI后線粒體破壞中的作用目的應(yīng)用Nrf2基因敲除小鼠來驗(yàn)證Nrf2通路在TBI后槲皮素對線粒體保護(hù)過程中的作用。方法選取野生型[Nrf(+/+)]小鼠與Nrf2基因敲除型[Nrf(-/-)]小鼠,采用相同的自由落體顱腦損傷模型。并在傷后30min給予腹腔注射槲皮素。在傷后24h取傷灶周圍大腦皮層組織使用Western blot檢測線粒體及胞漿中Bax及Cyt C的濃度。用酶標(biāo)儀檢測線粒體SOD及MDA濃度。結(jié)果與野生型小鼠相比,Nrf2基因敲除小鼠TBI后線粒體保護(hù)作用明顯減弱。結(jié)論Nrf2通路在槲皮素在TBI后線粒體保護(hù)中起重要作用。
[Abstract]:Background with the development of economy and society, traumatic brain injury (traumatic brain injury,TBI) has become one of the diseases with high mortality and disability rate. It has a certain degree of adverse effect on patients' physical and mental health, and also brings a serious burden to society. After TBI, the metabolism of brain tissue accelerates and the mitochondria in the brain tissue are injured. The change of function leads to the failure of normal energy metabolism, which leads to the breakdown of normal physiological function of cells, and finally leads to more serious secondary injury in the later stage [3]. During this process, a large number of mitochondria were destroyed in morphology and structure, resulting in the mitochondria unable to supply energy normally and resulting in the death of neuronal cells. Therefore, enhancing the biosynthesis of mitochondria to improve the energy metabolism of mitochondria can greatly reduce cell damage and protect neurons. Part one: quercetin could activate Nrf2 pathway after TBi and protect mitochondria from mitochondrial damage. Objective to investigate the protective effect of quercetin on mitochondrial damage induced by TBI through Nrf2 pathway. Methods male adult ICR mice were divided into four groups: sham operation group (sham group), trauma group (TBI group), trauma solvent group (TBI vehicle group) and traumatic quercetin group (TBI quercetin group). The expression of Nrf2 protein in nucleus and cytoplasm and the concentration of Bax and CytC in mitochondria and cytoplasm were detected by Western blot. The transfer of Nrf2 protein from cytoplasm to nucleus was detected by immunohistochemistry, and the concentration of SOD and MDA in mitochondria was detected by enzyme labeling instrument. Results compared with sham operation group, the expression of Nrf2 in trauma group and solvent group increased significantly. The contents of Cyt C and SOD in mitochondria decreased, while the contents of Bax and MDA increased. Quercetin not only promoted the entry and expression of Nrf2, but also decreased the expression of oxidative stress related proteins in mitochondria after traumatic brain injury. Conclusion Quercetin can further activate the Nrf2 signaling pathway after TBI and alleviate the mitochondrial damage after TBI. The second part of the role of Nrf2 pathway in reducing mitochondrial damage after TBI by quercetin objective to investigate the role of Nrf2 pathway in mitochondrial protection after TBI by using Nrf2 gene knockout mice. Methods Wild-type [Nrf (/)] mice and Nrf2 knockout type [Nrf (- / -) mice] were selected and the same free-fall brain injury model was used. Quercetin was injected intraperitoneally into 30min after injury. The concentrations of Bax and Cyt C in mitochondria and cytoplasm were detected by Western blot in the cortical tissue around the lesion 24 hours after injury. The concentrations of mitochondrial SOD and MDA were detected by enzyme labeling instrument. Results compared with wild-type mice, the mitochondrial protection of Nrf2 knockout mice was significantly weakened after TBI was removed. Conclusion Nrf2 pathway plays an important role in the mitochondrial protection of quercetin after TBI.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.15
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本文編號：2385168