HIV-gp120对神经小胶质细胞中脑源性神经营养因子（BDNF）表达的影响及其机制研究

发布时间：2018-01-28 21:00

  本文关键词： 脑源性神经营养因子 小胶质细胞 HIV-gp120 Wnt经典信号通路　出处：《浙江理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：艾滋病是对人类健康威胁最为严重的一种疾病,HIV则是致病的病原体。HIV-1的膜糖蛋白gp120和gp41在其感染过程中发挥着重要作用,在病毒进入细胞并与之相互作用时,gp120是一种典型的HIV毒性蛋白,对神经细胞具有毒性。本课题研究HIV-gp120对神经小胶质细胞BV2中脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)表达的影响和可能的机制。首先,MTT实验确定gp120作用于小胶质细胞BV2的合适浓度。与对照相比,用2ng/m L,10 ng/mL和50 ng/m L的gp120处理12 h后细胞的活力分别为80.1%,79.7%和80.5%。而剂量为1000 ng/m L的gp120使存活力降低至29.2%。因此,在后续实验中选择使用10 ng/m L的剂量,使其对细胞仅产生适当的毒性作用。其次,HIV-gp120分别作用于BV2细胞1 h,3 h,6 h,9 h,12 h和24 h,收集细胞样品并提取胞内蛋白进行Western blotting检测,分析BDNF的表达情况。结果显示:与对照相比proBDNF和mBDNF蛋白在gp120刺激BV2细胞3 h后表达量最高,分别是对照组的1.9倍和2.2倍。除此还发现,经gp120处理后小胶质细胞BV2被激活,并且伴随着CD11b表达量的升高,其表达量是对照组的1.62倍。与此同时,gp120处理的BV2细胞内也出现了Wnt3a和β-catenin的积累,Wnt5a的表达量没有显著性变化。因此,推测gp120是通过Wnt/β-catenin经典信号通路途径激活BV2细胞,促进BDNF的表达。为进一步确认其激活机制,本研究用不同浓度的Wnt3a蛋白(25 ng/mL、50 ng/m L、100 ng/m L)对信号通路进行激活,用不同浓度的DKK1(10 ng/m L、50 ng/m L、100 ng/mL)和IWR-1(0.1μM、1μM、10μM)抑制经典信号通路来进行更深入的研究。结果表明,Wnt3a(100 ng/mL)处理BV2细胞,增加了β-catenin的表达,其表达量是对照组的24倍,表示经典Wnt信号通路的激活。有趣的是,Wnt3a孵育后的小胶质细胞中BDNF表达上调1.81倍。相反,DKK1(100 ng/mL)和IWR-1(10μM)处理的细胞中则明显抑制了BDNF的表达,使其表达量分别下降64.6%和79.8%。在探索出DKK1和IWR-1的最适浓度后,本实验继续探究DKK1或IWR-1是否可以抑制由gp120引起的BDNF表达量的升高。结果表明:单独用DKK1或IWR-1处理BV2细胞组与gp120处理细胞组相比BDNF的表达量分别降低68.6%和62.6%。DKK1和gp120同时处理组或IWR-1和gp120同时处理组,BDNF的表达量与gp120处理细胞组相比分别降低70%和72.6%。此结果表明,DKK1和IWR-1可以显著性抑制由gp120引起的BDNF表达量的上调,说明阻断Wnt/β-catenin经典信号通路可以抑制BDNF的表达。进一步用DKK1阻断小胶质细胞BV2中的经典信号通路,探究DKK1对Wnt3a引起BDNF表达上调的影响。实验发现:在DKK1和Wnt3a处理的细胞组中BDNF的表达量与只用Wnt3a处理的细胞组相比下降73%,但与对照相比,其表达量依旧有所升高。此实验说明,DKK1可以抑制由Wnt3a引起的BDNF表达的升高,并且效果显著。进一步证明阻断Wnt/β-catenin经典信号通路可以抑制BDNF的表达。上述研究结果表明:HIV-gp120可以激活BV2细胞,并通过Wnt/β-catenin经典信号通路上调BDNF的表达。
[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.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91;R747.9

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