本文選題：HIV + NRTI　； 參考：《浙江理工大學》2017年碩士論文

【摘要】：核苷類逆轉錄酶抑制劑(NRTI)是一類重要的抑制人類免疫缺陷病毒(HIV)的抗病毒藥物,在高效抗逆轉錄病毒治療(HAART)中發(fā)揮關鍵作用,而AZT是HAART中應用最廣泛的藥物之一,被推薦為抗逆轉錄病毒藥物(ARVs)的首選藥物。雖然可以有效抑制HIV,但長期的HAART會導致HIV相關神經障礙(HAND)。有報道稱HAART導致的不良反應很大程度與神經炎癥有關。因此,目前急需尋找新的治療策略作為HAART的輔助療法來降低其引起的神經炎癥。據(jù)報道黃酮類化合物槲皮素有很好的抗炎效果,能夠減輕神經炎癥的癥狀,但是,槲皮素對NRTI引起的神經炎癥的影響及其機制尚未見報道,因此,本課題在這方面進行了系統(tǒng)的探索研究。首先進行體外實驗:通過Western blotting檢測不同濃度AZT(15,25,50μM)作用BV2細胞24 h后炎癥因子的表達情況,以及不同濃度槲皮素(10,20,30μM)預處理后,再以50μM AZT作用BV2細胞24 h后的炎癥因子表達情況。結果發(fā)現(xiàn)AZT藥物顯著增加BV2細胞炎癥因子(IL-1β,IL-6)釋放,與AZT組相比,濃度為15,25,50μM的槲皮素對BV2細胞中AZT誘發(fā)的IL-1β的抑制率分別是32.07±6.59%,27.75±6.92%,35.44±17.28%;對BV2細胞中IL-6的抑制率分別是29.80±1.49%,66.11±5.65%,68.63±14.27%。因此,槲皮素可有效降低AZT引起的炎癥因子釋放。體外實驗的探究初步證實槲皮素對NRTI藥物AZT導致的炎癥因子釋放具有顯著的抑制效果。其次進行體內實驗:以100mg/kg/day槲皮素和100mg/kg/dayAZT處理小鼠,8天后收集小鼠中樞神經組織(CNS:皮層,海馬,脊髓)。通過Western blotting檢測小鼠中樞神經組織(CNS)中炎癥因子(IL-1β,IL-6)的表達。結果發(fā)現(xiàn)槲皮素對小鼠中樞神經系統(tǒng)(CNS:皮層,海馬,脊髓)中AZT誘發(fā)的IL-1β的抑制率分別為35.02±3.61%,16.59±5.58%,20.78±6.15%;槲皮素對小鼠中樞神經系統(tǒng)(CNS:皮層,海馬,脊髓)中AZT誘發(fā)的IL-6的抑制率分別為39.43±14.21%,20.87±7.62%,17.67±1.17%。因此,槲皮素可以顯著降低小鼠CNS中AZT導致的炎癥因子上調。說明槲皮素可以有效抑制小鼠CNS中NRTI誘發(fā)的神經炎癥。神經炎癥以膠質細胞活化為標志,分別以GFAP和CD11b來標記星形膠質細胞和小膠質細胞,通過Western blotting和免疫組織化學染色的方法,進一步探究槲皮素對NRTI誘發(fā)的神經炎癥的效果。結果發(fā)現(xiàn)槲皮素可以有效抑制小鼠CNS中AZT引起的星型膠質細胞活化,以及脊髓和海馬組織中部分小膠質細胞的活化。說明槲皮素主要是通過抑制星型膠質細胞活化來抑制NRTI導致的神經炎癥,而對小膠質細胞的活化有部分抑制。從而進一步證明了槲皮素對NRTI導致的神經炎癥有抑制作用。上述實驗初步證實槲皮素可以抑制NRTI導致的神經炎癥,但其作用機制仍不太清楚。而Wnt5a信號通路與很多炎性疾病相關,并且最近很多研究發(fā)現(xiàn)Wnt5a信號通路參與神經炎癥調節(jié),因此我們猜測Wnt5a信號與槲皮素對NRTI導致的神經炎癥的抑制作用有關。因此,我們進行體內實驗和體外實驗通過Western blotting方法檢測Wnt5a表達水平的變化。結果發(fā)現(xiàn)AZT處理組Wnt5a顯著上調,而槲皮素可有效抑制Wnt5a的上調,說明Wnt5a參與調節(jié)槲皮素抑制NRTI藥物AZT導致的神經炎癥。因此,通過本課題的系統(tǒng)性研究證明槲皮素可以有效抑制膠質細胞活化進而降低NRTI導致的神經炎癥,并且Wnt5a細胞信號通路參與調節(jié)槲皮素對NRTI藥物AZT誘發(fā)的神經炎癥過程。
[Abstract]:Nucleoside reverse transcriptase inhibitor (NRTI) is an important class of antiviral drugs that inhibit human immunodeficiency virus (HIV) and plays a key role in high performance antiretroviral therapy (HAART). AZT is one of the most widely used drugs in HAART and is recommended as the first drug of the anti retroviral drug (ARVs). Although it can be effectively suppressed, it can be effectively suppressed. HIV, but long-term HAART can lead to HIV related neurological disorders (HAND). It is reported that the adverse reactions caused by HAART are largely associated with neuroinflammation. Therefore, it is urgent to find a new treatment strategy as a adjuvant therapy for HAART to reduce the neuroinflammation caused by it. It is reported that the flavonoid compound quercetin has good anti-inflammatory effects. It can alleviate the symptoms of neuro inflammation, but the effect of quercetin on NRTI induced neuroinflammation and its mechanism have not yet been reported. Therefore, this topic has been systematically explored in this field. First of all, in vitro experiment: the expression of inflammatory factors after 24 h of BV2 cells with different concentrations of AZT (15,25,50 uh M) was detected by Western blotting As well as the expression of inflammatory factors after the pretreatment of quercetin (10,20,30 mu M) at different concentrations (10,20,30 mu M), and after 24 h of BV2 cells with 50 micron AZT, it was found that AZT drugs significantly increased the release of BV2 cell inflammatory factors (IL-1 beta, IL-6), and the inhibitory rate of quercetin, which was the concentration of 15,25,50 micron, was compared with AZT group. It is 32.07 + 6.59%, 27.75 + 6.92%, 35.44 + 17.28%, and the inhibition rate of IL-6 in BV2 cells is 29.80 + 1.49%, 66.11 + 5.65%, 68.63 + 14.27%., so quercetin can effectively reduce the release of inflammatory factors caused by AZT. In vitro experiments have preliminarily confirmed that quercetin has a significant inhibitory effect on the release of inflammatory factors caused by NRTI drug AZT. Second, in vivo experiment: the mice were treated with 100mg/kg/day quercetin and 100mg/kg/dayAZT, and the mice central nervous tissue (CNS: cortex, hippocampus, spinal cord) were collected 8 days later. The expression of inflammatory factors (IL-1 beta, IL-6) in the central nervous tissue (CNS) of mice was detected by Western blotting. The results showed that quercetin was used in the central nervous system of mice (CNS: cortex, CNS: cortex,). The inhibitory rates of AZT induced IL-1 beta in the hippocampus and spinal cord were 35.02 + 3.61%, 16.59 + 5.58%, 20.78 + 6.15%, respectively. The inhibition rate of AZT induced IL-6 in the central nervous system (CNS: cortex, hippocampus, and spinal cord) of mice was 39.43 + 14.21%, 20.87 + 7.62%, 17.67 + 1.17%., respectively. Quercetin could significantly reduce AZT induced inflammation in CNS in mice. It is suggested that quercetin can effectively inhibit NRTI induced neuroinflammation in CNS of mice. Neuroglia is marked by glial cell activation, and astrocytes and microglia are marked with GFAP and CD11b respectively. The NRTI induced God by quercetin is further explored by Western blotting and immunohistochemical staining. The results showed that quercetin could effectively inhibit the activation of astrocytes induced by AZT in CNS and the activation of some microglia in the spinal and hippocampal tissues. It shows that quercetin mainly inhibits the activation of astrocytes by inhibiting the activation of NRTI, and has a part of the activation of microglia. The inhibitory effect of quercetin on NRTI induced neuroinflammation was further demonstrated. The above experiments preliminarily confirmed that quercetin inhibited NRTI induced neuroinflammation, but its mechanism was still unclear. The Wnt5a signaling pathway was associated with many inflammatory diseases, and the most recent studies found that Wnt5a signaling pathways involved nerves. We speculated that the Wnt5a signal and quercetin were associated with the inhibitory effect of quercetin on NRTI induced neuroinflammation. Therefore, in our in vivo and in vitro experiments, the expression of Wnt5a was detected by the Western blotting method. The results showed that the Wnt5a in the AZT treatment group was significantly adjusted, and quercetin could effectively inhibit the up regulation of Wnt5a. Wnt5a is involved in regulating quercetin to inhibit the neuroinflammation caused by the NRTI drug AZT. Therefore, the systematic study of this topic has demonstrated that quercetin can effectively inhibit glial activation and reduce the neuroinflammation caused by NRTI, and the Wnt5a cell signaling pathway participates in the regulation of quercetin on the process of neuroinflammation induced by NRTI drug AZT.
【學位授予單位】：浙江理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96

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