本文關(guān)鍵詞： 腦源性神經(jīng)營養(yǎng)因子 小膠質(zhì)細胞 HIV-gp120 Wnt經(jīng)典信號通路　出處：《浙江理工大學》2017年碩士論文　論文類型：學位論文

【摘要】：艾滋病是對人類健康威脅最為嚴重的一種疾病,HIV則是致病的病原體。HIV-1的膜糖蛋白gp120和gp41在其感染過程中發(fā)揮著重要作用,在病毒進入細胞并與之相互作用時,gp120是一種典型的HIV毒性蛋白,對神經(jīng)細胞具有毒性。本課題研究HIV-gp120對神經(jīng)小膠質(zhì)細胞BV2中腦源性神經(jīng)營養(yǎng)因子(brain-derived neurotrophic factor,BDNF)表達的影響和可能的機制。首先,MTT實驗確定gp120作用于小膠質(zhì)細胞BV2的合適濃度。與對照相比,用2ng/m L,10 ng/mL和50 ng/m L的gp120處理12 h后細胞的活力分別為80.1%,79.7%和80.5%。而劑量為1000 ng/m L的gp120使存活力降低至29.2%。因此,在后續(xù)實驗中選擇使用10 ng/m L的劑量,使其對細胞僅產(chǎn)生適當?shù)亩拘宰饔谩Ｆ浯?HIV-gp120分別作用于BV2細胞1 h,3 h,6 h,9 h,12 h和24 h,收集細胞樣品并提取胞內(nèi)蛋白進行Western blotting檢測,分析BDNF的表達情況。結(jié)果顯示:與對照相比proBDNF和mBDNF蛋白在gp120刺激BV2細胞3 h后表達量最高,分別是對照組的1.9倍和2.2倍。除此還發(fā)現(xiàn),經(jīng)gp120處理后小膠質(zhì)細胞BV2被激活,并且伴隨著CD11b表達量的升高,其表達量是對照組的1.62倍。與此同時,gp120處理的BV2細胞內(nèi)也出現(xiàn)了Wnt3a和β-catenin的積累,Wnt5a的表達量沒有顯著性變化。因此,推測gp120是通過Wnt/β-catenin經(jīng)典信號通路途徑激活BV2細胞,促進BDNF的表達。為進一步確認其激活機制,本研究用不同濃度的Wnt3a蛋白(25 ng/mL、50 ng/m L、100 ng/m L)對信號通路進行激活,用不同濃度的DKK1(10 ng/m L、50 ng/m L、100 ng/mL)和IWR-1(0.1μM、1μM、10μM)抑制經(jīng)典信號通路來進行更深入的研究。結(jié)果表明,Wnt3a(100 ng/mL)處理BV2細胞,增加了β-catenin的表達,其表達量是對照組的24倍,表示經(jīng)典Wnt信號通路的激活。有趣的是,Wnt3a孵育后的小膠質(zhì)細胞中BDNF表達上調(diào)1.81倍。相反,DKK1(100 ng/mL)和IWR-1(10μM)處理的細胞中則明顯抑制了BDNF的表達,使其表達量分別下降64.6%和79.8%。在探索出DKK1和IWR-1的最適濃度后,本實驗繼續(xù)探究DKK1或IWR-1是否可以抑制由gp120引起的BDNF表達量的升高。結(jié)果表明:單獨用DKK1或IWR-1處理BV2細胞組與gp120處理細胞組相比BDNF的表達量分別降低68.6%和62.6%。DKK1和gp120同時處理組或IWR-1和gp120同時處理組,BDNF的表達量與gp120處理細胞組相比分別降低70%和72.6%。此結(jié)果表明,DKK1和IWR-1可以顯著性抑制由gp120引起的BDNF表達量的上調(diào),說明阻斷Wnt/β-catenin經(jīng)典信號通路可以抑制BDNF的表達。進一步用DKK1阻斷小膠質(zhì)細胞BV2中的經(jīng)典信號通路,探究DKK1對Wnt3a引起B(yǎng)DNF表達上調(diào)的影響。實驗發(fā)現(xiàn):在DKK1和Wnt3a處理的細胞組中BDNF的表達量與只用Wnt3a處理的細胞組相比下降73%,但與對照相比,其表達量依舊有所升高。此實驗說明,DKK1可以抑制由Wnt3a引起的BDNF表達的升高,并且效果顯著。進一步證明阻斷Wnt/β-catenin經(jīng)典信號通路可以抑制BDNF的表達。上述研究結(jié)果表明:HIV-gp120可以激活BV2細胞,并通過Wnt/β-catenin經(jīng)典信號通路上調(diào)BDNF的表達。
[Abstract]:HIV is the most serious threat to human health. HIV is the pathogenic pathogen. HIV-1 membrane glycoprotein gp120 and gp41 play an important role in the process of infection. Gp120 is a typical HIV toxic protein when it enters cells and interacts with it. In this study, we studied the effects of HIV-gp120 on brain-derived neurotrophic factor (brain-derived neurotrophic factor) in microglial BV2. Brain-derived neurotrophic factor. The effect of BDNF on the expression of BDNF and its possible mechanism. Firstly, the appropriate concentration of gp120 on BV2 of microglia cells was determined by MTT assay. Compared with the control group, 2ng / mL was used. The viability of cells treated with 10 ng/mL and 50 ng/mL gp120 for 12 h was 80.1%, respectively. 79.7% and 80.5 doses of gp120 reduced the survivability to 29.2g / L. Therefore, 1 000 ng/m / L gp120 reduced the viability to 29. 2%. In the subsequent experiments, 10 ng/m / L dose was used to make the cells only have a proper toxic effect. Secondly, HIV-gp120 was applied to BV2 cells for 1 h or 3 h respectively. The cell samples were collected and the intracellular proteins were extracted for Western blotting detection. The expression of BDNF was analyzed. The results showed that the expression of proBDNF and mBDNF protein was the highest in BV2 cells stimulated by gp120 for 3 h compared with the control group. In addition, the microglia BV2 was activated after gp120 treatment and accompanied by the increase of CD11b expression. At the same time, the accumulation of Wnt3a and 尾 -catenin was also found in the BV2 cells treated with gp120. There was no significant change in the expression of Wnt5a. Therefore, it is assumed that gp120 activates BV2 cells through WNT / 尾 -catenin classical signaling pathway. To further confirm the activation mechanism of BDNF, we used different concentrations of Wnt3a protein 25 ng / mL ~ 50 ng/mL. 100 ng/m L) was used to activate the signaling pathway with different concentrations of DKK1(10 ng/m L ~ (50 ng/m / L). 100ng / mL and IWR-1(0.1 渭 MN 1 渭 MN 10 渭 M) inhibited the classical signal pathway for further study. The expression of 尾 -catenin was increased by the treatment of Wnt3a(100 ng / mL, and the expression of 尾 -catenin was 24 times higher than that of the control group. It is interesting that the expression of BDNF in microglia treated with Wnt3a is increased by 1.81 times. DKK1(100 ngmL) and IWR-1(10 渭 M) inhibited the expression of BDNF. The expression of DKK1 and IWR-1 were decreased by 64.6% and 79.8, respectively. The present study continued to explore whether DKK1 or IWR-1 could inhibit the increase of BDNF expression induced by gp120. The results showed that:. The expression of BDNF in BV2 cells treated with DKK1 or IWR-1 alone was decreased by 68.6% and 62.6%, respectively, compared with that in gp120 treated cells group. DKK1 and gp120 were also decreased at the same time. Processing groups or IWR-1 and gp120 groups at the same time. The expression of BDNF was decreased by 70% and 72.6, respectively, compared with that of gp120 treated cells. DKK1 and IWR-1 could significantly inhibit the up-regulation of BDNF expression induced by gp120. It is suggested that blocking Wnt- 尾 -catenin signal pathway can inhibit the expression of BDNF, and further block the classical signal pathway in microglia BV2 by DKK1. To explore the effect of DKK1 on the upregulation of BDNF expression induced by Wnt3a. The expression of BDNF in cells treated with DKK1 and Wnt3a decreased by 73% compared with that of cells treated only with Wnt3a. However, the expression of DKK1 was still higher than that of the control. This experiment indicated that DKK1 could inhibit the increase of BDNF expression induced by Wnt3a. The results showed that blocking Wnt- 尾 -catenin signal pathway could inhibit the expression of BDNF. HIV-gp120 can activate BV2 cells. The expression of BDNF was up-regulated by Wnt- 尾 -catenin signal pathway.
【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】：R512.91;R747.9

【參考文獻】

相關(guān)期刊論文 前3條

1 陳大偉;劉燕青;張朝;;腦源性神經(jīng)營養(yǎng)因子的研究進展[J];北方藥學;2015年03期

2 鄭向紅;許穎;;神經(jīng)膠質(zhì)細胞作用的研究進展[J];海峽藥學;2013年03期

3 談丹丹;洪道俊;徐仁O5;吳裕臣;;神經(jīng)膠質(zhì)細胞和神經(jīng)退行性疾病[J];中國老年學雜志;2013年02期

，

本文編號：1471552