【作者】 李晓光；

【导师】 王奇；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2016， 博士

【摘要】 目的:一、通过观察中药单体成分白藜芦醇对BV-2小胶质细胞A β吞噬作用和炎症因子分泌的影响,及其对Trem2蛋白表达及分泌的调节作用,探讨白藜芦醇作为抗AD药物研发的可能性。二、通过观察过表达PS1对BV-2小胶质细胞吞噬作用的影响及其对Trem2转运的调控,探讨以PS1为间接作用靶点调控Trem2的转运,增强小胶质细胞对A β的吞噬,为抗AD中药的筛选提供更多的思路。方法:一、白藜芦醇对BV-2细胞吞噬作用的调控及其机制研究1.CCK-8检测细胞活力:选取白藜芦醇5μM、10μM、20μM、40μM为干预浓度,24小时或48小时后,加入CCK-8试剂,37℃C孵育2小时,在酶标仪中492nm处读取96孔板吸光值。2.流式细胞术:对BV-2细胞进行不同的干预之后,加入荧光标记的A β孵育2小时,流式细胞仪检测细胞荧光强度来测定BV-2细胞对于Aβ的吞噬作用。3.分子生物学实验:对BV-2细胞进行不同的干预之后,采用Western blot检测BV-2细胞Trem2总蛋白表达及上清中sTrem2的分泌量,采用Real-time PCR检测细胞IL-1 β、TNF-α的mRNA转录水平。二、PS1对BV-2细胞吞噬作用的调控及其机制研究1.分子生物学实验:用免疫共沉淀方法检测PS1与Trem2的蛋白间相互作用,以细胞膜表面生物素化的方法检测过表达PS1对BV-2细胞内Trem2蛋白转运的影响。2.载体病毒感染:采用病毒感染的方法对构建BV-2细胞的稳定转染细胞系。3.流式细胞术:采用流式细胞术检测PS1过表达对于BV-2细胞A β吞噬作用的影响。结果:一、白藜芦醇对BV-2细胞吞噬作用的调控及其机制研究1.在干预24小时的条件下,5 μM、10 μM、20 μM浓度组白藜芦醇与各自的空白对照组比较,对于BV-2细胞活力的影响均无明显的差异(P>0.05),40μM浓度组可降低BV-2细胞的活力(P<0.05)。而在干预48小时的条件下,5μM、10μM浓度组对于BV-2细胞的活力仍然没有影响(P>0.005),20 μ M、40 μ M浓度组可降低BV-2细胞的活力(P<0.05)。2.5 μM的白藜芦醇对于BV-2细胞的吞噬作用略有促进,但相比于对照组,差异不具有统计学意义(P>0.05);10 μM、20 μM浓度组相对于对照组显著促进吞噬作用,并且相比于对照组具有统计学差异(P<0.05)。3.白藜芦醇10uM剂量组BV-2细胞的Trem2蛋白表达量略有升高,但与对照组比较尚不具有统计学差异(P>0.05);20μM剂量组可明显升高Trem2蛋白表达量,相对对照组表达量约为后者的1.7倍,且差异具有显著的统计学意义(P<0.01)。4.白藜芦醇10 μ M剂量组可一定程度上抑制小胶质细胞sTrem2的分泌,但与对照组比较,差异尚不具有统计学意义(P>0.05);20μM剂量组可明显抑制sTrem2的分泌,相对对照组的分泌量为后者的70%左右,且差异具有显著的统计学意义(P<0.01)。5.Trem2-Fc4nM、20nM剂量组与相应浓度Fc对照组比较,BV-2细胞对Aβ的吞噬作用略有降低,但无统计性差异(P>0.05);而40nM剂量组与相应浓度对照组比较,BV-2细胞对A β的吞噬作用明显降低,且差异具有统计学意义(P<0.01)。6.不同剂量的白藜芦醇干预炎症反应模型BV-2细胞后,白藜芦醇各个剂量组均可降低细胞IL-1β的mRNA水平,且差异均具有统计学意义(P<0.05)。白藜芦醇对TNF-α的mRNA转录具有一定的下调作用,但是各剂量组相对对照组差异不具有统计学意义(P>0.05)。二、PS1对BV-2细胞吞噬作用的调控及其机制研究1.Trem2可以共沉淀PS1全长蛋白、PS1-NTF和PS1-CTF,而IgG对照组并没有检测到相应蛋白条带。反向的验证也证明,PS1-NTF抗体与PS1-CTF抗体的免疫沉淀物中,均可检测到Trem2的表达,而IgG对照组并没有出现相应的蛋白条带。2.BV-2-Vector组与BV-2-PS1组Trem2的蛋白总表达量基本一致,不存在差异性。细胞膜Trem2与总Trem2比值的结果分析表明,BV-2-PS1组比值相对BV-2-Vector组明显下降,且该差异具有统计学意义(P<0.05)。3.BV-2-PS1细胞系对于FAM-A β的吞噬明显低于BV-2-Vector细胞系,且差异具有统计学意义(P<0.05)。BV-2-PS1-Trem2细胞系相比BV-2-PS1细胞系对于FAM-Aβ的吞噬明显增加,且差异具有统计学意义(P<0.05)。结论:一、白藜芦醇对BV-2细胞吞噬作用的调控及其机制研究1.白藜芦醇干预可以促进BV-2细胞的吞噬功能,并且该促进作用存在剂量依赖性。2.白藜芦醇可上调细胞Trem2总蛋白的表达,下调sTrem2的分泌。3.白藜芦醇调节BV-2细胞吞噬功能的可能机制为上调细胞Trem2的表达和下调sTrem2的分泌。4.白藜芦醇可显著降低A β诱导的BV-2细胞IL-1 β的分泌,对TNF-α的分泌也有一定的下调作用,但结果无统计学意义,需要进一步探讨。二、PS1对BV-2细胞吞噬作用的调控及其机制研究1.PS1与Trem2之间存在蛋白间相互作用。2.过表达PS1可减少BV-2细胞的Trem2膜转运。3.过表达PS1可降低BV-2细胞的吞噬,在此基础上进一步过表达Trem2可恢复吞噬功能至基线水平,说明PS1降低BV-2细胞吞噬功能的机制为减少了 Trem2的膜转运。更多还原

【Abstract】 ObjectiveAim 1:by the study on the effect of resveratrol on the phagocytosis of A β,the inflammatory cytokine secretion and the regulation of protein expression of Trem2,we would like to discover the potential of resveratrol to be an anti-AD drug.Aim 2:,by the study on the effect of PS1 overexpression on phagocytosis of A β and the regulation of Trem2 trafficking,we provide a new possible way to the screening of Trem2-targeted traditional herbs.Methods1.The effect of resveratrol on phagocytosis function of BV-2 cell line and the underlying mechanisms1.1 CCK-8 cell viability:Select 5 μ M,10 μ M,20 μ M and 40 μ M as treating concentrations.24 hours or 48 hours later,add CCK-8 reagent and incubated for 2 hours at 37 ℃.Then read the absorbance values at 492nm.1.2 Flow cytometry:Treat BV-2 cell line with different concentrations of resveratrol or sTrem2 for 24 hours.Then incubate with fluorescent labeled Aβ for 2 hours,followed by flow cytometry to detect the phagocytosis of Aβ.1.3 Molecular biology experiments:Treated with different concentrations of resveratrol,Trem2 expression and sTrem2 secretion of BV-2 cells were detected by Westerm Blot.IL-1 β and TNF-α mRNA transcript levels was detected by real-time PCR.2.The effect of PS1 on phagocytosis function of BV-2 cell line and the underlying mechanisms2.1 Molecular biology experiment:Detect the interaction between PS1 and Trem2 by co-immunoprecipitation,and study the effect of PS1 overexpression on Trem2 trafficking by cell surface biotinylation.2.2 Viral vector infection:Screen PS1/Trem2 overexpression stable BV-2 cell lines.2.3 Flow cytometry:Flow cytometry was used to detect the effect of PS1 overexpression on phagocytosis of BV-2 cell line.Results1.The effect of resveratrol on phagocytosis function of BV-2 cell and the underlying mechanisms1.1 With the treatment of 5 u M,10 u M or 20 u M of resveratrol for 24h,there is no effect on cell viability compared with the control groups respectively,while 40 μM resveratrol can damage the cell viability of BV-2 cell line.With the treatment of 5 μ M or 10 μ M of resveratrol for 48h,there is no effect on cell viability compared with the control groups respectively,while 20 μM and 40 μ M resveratrol can damage the cell viability of BV-2 cell line.1.2 5μM resveratrol increased BV-2 phagocytosis slightly.But compared to the control group,the difference was not statistically significance;10 μ M and 20 u M resveratrol significantly increased BV-2 phagocytosis compared with control group.1.3 10μM resveratrol increases Trem2 expression slightly,but there is no statistical difference compared with the control group(P>0.05);20 μ M resveratrol significantly increases the expression of Trem2,the expression of which is about 1.7 times relative to the control group,and there is statistically difference between the two groups(P<0.01).1.4 10 u M resveratrol may reduce sTrem2 secretion,but there is no statistical difference compared with the control group(P>0.05);20 u M resveratrol significantly reduces sTrem2 secretion,the expression of which is about 70%of the control group,(P<0.01).1.5 4nM and 20nM Trem2-Fc reduce phagocytosis of Aβ slightly,but there is no statistical difference compared with the control group(P>0.05).40nM dose group reduces the phagocytosis of A β,and the difference is statistically significant(P<0.01).1.6 With the treatment of different doses of resveratrol,the mRNA levels of IL-1β are reduced in BV-2 cell line,and the differences are statistically significant(P<0.05).5μM dose resveratrol do not affect the TNF-α mRNA transcription,but resveratrol 10 u M and 20 u M group,mRNA expression level of TNF-a are decreased,but the decreases are not statistically significant(P>0.05).2.The effect of Presenilin-1 on phagocytosis function of BV-2 cell and the underlying mechanisms1.Trem2 full-length protein can be co-precipitated with PS1(PS1 full lenth,PS1-FL),PS1-NTF and PS1-CTF,but can not detect the expression of NCT,and the protein band is not detected in the IgG control group.As verified by the reverse experiments,Trem2 expression can be detected in the immunoprecipitates of PS1-NTF antibody(Ab14)and PS1-CTF antibodies(Anti PS1-loop),and the control IgG group does not show a corresponding protein bands band.2.The Term2 overall expression level in BV-2-Vector Group is consistent with that of BV-2-PS1 group,without significant difference.Results from Surface Trem2 and Total Trem2 expression analysis show that the Surface/Total Trem2 ratio in BV-2 overexpressed PS1 group is significantly decreased and the difference is statistically significant(P<0.05).3.The phagocysis effect of BV-2-PS1 cell line on FAM-Aβ is significantly lower than that of BV-2-Vector cell lines,and the difference is statistically significant(P<0.05).Compared to the BV-2-PS1 cell line,the phagocysis effect of BV-2-PS1-Trem2 on FAM-Aβ is significantly increased,and the difference is statistically significant(P<0.05).Conclusion1.The effect of resveratrol on phagocytosis function of BV-2 cell and the underlying mechanisms.1.1 Resveratrol interventions can enhance microglial phagocytosis,and enhancement shows dose-dependent effect.1.2 Resveratrol can upregulate the overall protein expression of Trem2 and downregulate the secretion of sTrem2.1.3 The mechanisms underlying the regulation of resveratrol on the BV-2 microglial phagocytosis possibly involve the upregulation of total Trem2 and downregulation of sTrem2 secretion.1.4 Resveratrol can significantly reduce the secretion level of IL-1β from Aβ-induced BV-2 microglial cells and has downward effect on the secretion of TNF-α.However,the results are not statistically significant,which require further exploration.2.Regulation and mechanism of PS1 within the BV-2 microglia endocytosis 2.1 Protein interactions exist between PS1 and Trem2.2.2 Overexpression of PS1 can reduce the membrane transcportation of Trem2 in BV-2 microglial cells.2.3 Overexpression of PS1 decreases the phagocytosis function of BV-2 microglia.Based on this,overexpression of Trem2 can restore the phagocytosis to the baseline level.This suggests that PS1 reduced microglia phagocytosis may be induced by the reduction of Term2 membrane transportation.更多还原