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衰老相关性空间学习和记忆能力减退是常见的生物学现象,且易受到许多因素的加速,但发病机制尚不完全明确。现已证明海马介导空间学习和记忆,且容易受衰老的影响[1]。海马突触可塑性的改变是衰老相关性学习记忆减退的基础[2]。研究[3]表明神经营养因子,如脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)是突触可塑性的关键调控因子,参与了记忆的形成,而BDNF基因的表达会随着年龄的增长而下降,进而涉及老年期认知损害[4]。证据[5-6]表明胚胎期的不良因素暴露如炎症、铅、营养不良可加速年龄相关性认知损害。我们前期的研究[7]表明,母鼠孕晚期暴露脂多糖(LPS)诱导的炎症会加重子代小鼠中老年期学习和记忆损害。然而,胚胎期炎症暴露是否影响中年期海马BDNF基因表达尚不清楚。
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SPF级2月龄CD-1系小白鼠,雌鼠20只,雄鼠10只,购于北京维通利华责任有限公司。所有小鼠均在标准实验室条件下饲养[室温为(23±1)℃,湿度为55%±5%,明暗周期为12 h,可随意饮食和水]。适应2周后,将雌雄鼠以2∶ 1的比例合笼,次日清晨检查到阴栓者为受孕第0天。在母鼠怀孕第15~17天腹腔注射LPS(50 μg/kg)或等量的0.9%氯化钠溶液,在出生后第21天,将子鼠与母鼠分开,每笼3~4只同性别子鼠饲养。对应子鼠分别为CON组和LPS组,每组40只,雌雄各半,分别饲养至3月龄、15月龄时,完成行为学实验,随后每组随机抽取6只进行Western blotting和实时定量PCR实验。其中有任何运动障碍、脱毛或可见肿瘤的动物在实验前都被排除在外。所有实验遵从安徽医科大学动物伦理委员会要求(No.LLSC20160165)。
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采用Morris水迷宫实验检测小鼠的学习和记忆功能,该行为学测试在直径150 cm、深度30 cm的黑色圆形不锈钢水池中进行,池中充满水(25 ℃),水深25 cm,分为四个等量的象限(Ⅰ、Ⅱ、Ⅲ和Ⅳ),圆柱形逃生平台(直径10 cm)隐藏在第一象限中心的水下1 cm处,水迷宫周围有一层不透明的白色窗帘,在距水面1 m的位置悬挂3个不同的黑色标志物,来帮助小鼠获取位置信息。该实验分为两个阶段:定位航行期(学习期)和空间探索期(记忆期)。将小鼠从不同象限且面向侧壁入水,允许其自由游动60 s,若找到逃生平台,则允许该小鼠在平台上停留30 s,然后开始下一次实验。未能在60 s内找到平台的小鼠从水池中被救出,并在下一次实验开始前在平台上放置30 s。每天4次,每次间隔15 min,连续7 d。待第7天学习期结束后,开始进行记忆期实验,撤去平台,将小鼠从平台相反的象限开始,自由探索60 s。Any-maze软件记录小鼠找到平台的游泳路程(代表学习能力)以及小鼠在靶象限(平台所在象限)内的游泳路程占总路程的百分比(代表记忆能力)。
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Morris水迷宫任务完成后第15天,将小鼠颈椎脱臼处死迅速取脑,冰上取海马组织后放置-80 ℃冰箱冷冻保存。
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将海马组织解冻后,将其充分研磨,加入适当的RIPA细胞裂解液,高速离心,裂解组织,收集上清液。往上清液中按1∶ 4比例加入SDS-PAGE蛋白上样缓冲液。水浴(100 ℃)加热10 min,使组织充分变性,待样品冷却至室温后,直接加到SDS-PAGE胶加样孔内,浓缩胶100 V,时间为30 min;分离胶电压100 V,时间为60 min。恒流转模后,继续漂洗5 min,加入Western封闭液,室温条件下封闭2 h,加入一抗兔抗-BDNF(1∶ 1 000, ab108319,美国Abcam公司), 4 ℃孵育过夜后使用PBST洗涤。加入二抗羊抗兔IgG (1∶ 20 000,Zsbio,ZB2301),室温孵育2 h后继续PBST洗涤,随后用发光试剂盒来检测蛋白。使用Image J软件对蛋白条带的结果进行分析,计算BDNF的相对表达量。
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称取海马组织50 mg,剪碎,液态研磨,加入1 mL TRIzol进行裂解,提取RNA组织,并检测其纯度。将RNA经RT反应,逆转录生成cDNA,取cDNA作为荧光定量的模板。反应体系如下, 5 μL 2× SYBR Green Mixture、1 μL Forward primer(10 μmol/L)、1 μL Reverse primer (10 μmol/L)、1 μL cDNA、2 μL RNase Free water。反应条件如下, Hold(预变性):95 ℃、60 s、单循环;PCR反应:95 ℃、20 s,60 ℃、60 s,共40循环。本次实验所使用的计算方法为2-△△Ct。引物序列见表 1。
Target genes Forward primer(5′-3′) Reverse primer(5′-3′) β-actin AGT GTG ACG TTG ACA TCC GT TGC TAG GAG CCA GAG CAG TA BDNF TTA CTC TCC TGG GTT CCT GA ACG TCC ACT TCT GTT TCC TT 表 1 引物序列
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所有数据均使用SPSS25进行统计分析,所有资料均符合正态分布。Morris水迷宫成绩用重复测定方差分析及双因素方差分析进行统计。其中,组间数据分析采用LSD检验。海马BDNF基因表达水平与Morris水迷宫结果之间的相关性用Pearson相关进行分析。
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CON组小鼠游泳路程随时间的增加而逐渐降低[F(6,216) = 35.968,P < 0.01]。15月龄比3月龄CON组游泳路程明显延长[F(1,36)=10.949,P < 0.01],但性别及性别与年龄的交互效应均无统计学意义(P>0.05)(见图 1A)。在3月和15月龄,LPS的处理效应均显著[F(1,36)=6.016,P < 0.01;F(1,36)=9.925,P < 0.01],即在两个年龄段,LPS组的游泳路程均明显长于CON组,但性别及性别与处理的交互效应均无统计学意义(P>0.05)(见图 1B、C)。
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15月龄CON组小鼠靶象限游泳路程百分比明显低于3月龄CON组[F(1,36) =16.950,P < 0.01],但两个年龄段性别差异均无统计学意义(P>0.05)。在3月龄和15月龄,LPS组均比CON组小鼠靶象限游泳路程百分比均明显降低[F(1,36)=8.139,P < 0.01;F(1,36) =17.480,P < 0.01],各组性别效应无统计学意义(P>0.05)(见图 1D)。2.3海马BDNF基因表达水平各组免疫印迹条带图见图 2A。在CON组中,15月龄较3月龄小鼠海马BDNF蛋白和mRNA相对含量均明显降低[F(1,20) = 203.300,P < 0.01;F(1,20)=86.430,P < 0.01],但性别效应无统计学意义(P>0.05)。在3月龄和15月龄,LPS组较CON组小鼠海马BDNF蛋白[F(1,23)=233.764,P < 0.01;F(1,23)=706.340,P < 0.01]和mRNA[F(1,23)=335.422,P < 0.01;F(1,23)=93.868,P < 0.01]相对含量均显著降低,各组性别效应无统计学意义(P>0.05)(见图 2B、C)。2.4小鼠空间学习记忆能力与海马BDNF基因表达水平的相关性Pearson分析表明,15月龄CON组和LPS组小鼠海马BDNF蛋白水平与平均游泳路程呈负相关关系(r=-0.649,P < 0.05;r=-0.871,P < 0.01),与靶象限游泳路程百分比呈正相关关系(r=0.780,P < 0.01;r=0.840,P < 0.01);小鼠海马BDNF mRNA相对含量与平均游泳路程呈负相关关系(r=-0.891,P < 0.01;r=-0.582,P < 0.05),与靶象限游泳路程百分比呈正相关关系(r=0.883,P < 0.01;r=0.712,P < 0.01)。3月龄海马BDNF蛋白和mRNA相对含量与平均游泳路程和靶象限游泳路程百分比均无明显相关关系(P>0.05)(见表 2)。
分组 Morris水迷宫成绩 BDNF BDNF mRNA 3月龄CON组 平均游泳路程 -0.574(0.051) -0.560(0.058) 靶象限游泳路程百分比 0.564(0.056) 0.554(0.062) 3月龄LPS组 平均游泳路程 -0.324(0.304) -0.182(0.571) 靶象限游泳路程百分比 0.116(0.720) 0.110(0.732) 15月龄CON组 平均游泳路程 -0.649(0.022)* -0.891(0.001)** 靶象限游泳路程百分比 0.780(0.003)** 0.883(0.001)** 15月龄LPS组 平均游泳路程 -0.871(0.001)** -0.582(0.047)** 靶象限游泳路程百分比 0.840(0.001)** 0.712(0.009)* *P < 0.05,**P < 0.01 表 2 海马BDNF基因表达水平与Morris水迷宫成绩的相关性(r)
胚胎期炎症暴露对中老年海马脑源性神经营养因子基因表达和认知功能的影响
Effects of embryonic inflammatory exposure on hippocampal BDNF gene expression and cognitive function in middle-aged and elderly mice
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摘要:
目的探讨胚胎期炎症暴露对中老年期小鼠海马脑源性神经营养因子(BDNF)蛋白和mRNA表达水平与认知功能的影响。 方法母鼠怀孕第15~17天腹腔注射脂多糖(LPS, 50 μg/kg)或等量的0.9%氯化钠溶液, 对应子鼠分别为LPS组和CON组(雌雄各半), 分别于3月龄、15月龄时完成Morris水迷宫任务, 用Western blotting和实时定量PCR检测海马BDNF表达水平。 结果与3月龄CON组相比, 15月龄CON组游泳路程明显延长(P < 0.01), 靶象限游泳路程百分比和海马BDNF基因表达水平均下降(P < 0.01)。在两个年龄段, LPS组Morris水迷宫成绩及BDNF基因表达水平均比CON组差。Pearson相关分析显示, 仅15月龄CON组和LPS组BDNF基因表达水平与游泳路程呈负相关关系(P < 0.05), 与靶象限游泳路程百分比呈正相关关系(P < 0.05)。 结论胚胎期炎症暴露可加重中老年小鼠空间学习记忆能力损害, 且可能与海马BDNF基因表达水平下降有关。 Abstract:ObjectiveTo explore the effects of embryonic inflammatory exposure on brain-derived neurotrophic factor (BDNF) protein and mRNA expression in hippocampus and cognitive function in middle-aged and elderly mice. MethodsFemale rats were intraperitoneally injected with lipopolysaccharide (LPS, 50 μg/kg) or 0.9% sodium chloride solution on the fifteenth to seventeenth day of pregnancy, and the corresponding offsprings were set as LPS group and CON group, with equal male and female mice in each group.The Morris water maze task was completed at 3 and 15 months of age, and the BDNF expression level in hippocampus was detected by Western blotting and real-time quantitative PCR. ResultsCompared with the CON group at 3 months, the swimming distance in the CON group at 15 months was significantly longer (P < 0.01), and the percentage of swimming distance in the target quadrant and the expression level of BDNF gene in hippocampus were decreased (P < 0.01).Morris water maze scores and BDNF expression levels in the LPS group were worse than those in the CON group in the both age groups.Pearson correlation analysis showed that BDNF gene expression level was negatively correlated with swimming distance in the CON group and LPS group at 15 months of age (P < 0.05), and positively correlated with percentage of swimming distance in the target quadrant (P < 0.05). ConclusionsEmbryonic inflammatory exposure can aggravate the spatial learning and memory ability of middle-aged and elderly mice, which may be related to the decreased expression of BDNF gene in hippocampus. -
Key words:
- aging /
- brain-derived neurotrophic factor /
- mice /
- learning and memory
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表 1 引物序列
Target genes Forward primer(5′-3′) Reverse primer(5′-3′) β-actin AGT GTG ACG TTG ACA TCC GT TGC TAG GAG CCA GAG CAG TA BDNF TTA CTC TCC TGG GTT CCT GA ACG TCC ACT TCT GTT TCC TT 表 2 海马BDNF基因表达水平与Morris水迷宫成绩的相关性(r)
分组 Morris水迷宫成绩 BDNF BDNF mRNA 3月龄CON组 平均游泳路程 -0.574(0.051) -0.560(0.058) 靶象限游泳路程百分比 0.564(0.056) 0.554(0.062) 3月龄LPS组 平均游泳路程 -0.324(0.304) -0.182(0.571) 靶象限游泳路程百分比 0.116(0.720) 0.110(0.732) 15月龄CON组 平均游泳路程 -0.649(0.022)* -0.891(0.001)** 靶象限游泳路程百分比 0.780(0.003)** 0.883(0.001)** 15月龄LPS组 平均游泳路程 -0.871(0.001)** -0.582(0.047)** 靶象限游泳路程百分比 0.840(0.001)** 0.712(0.009)* *P < 0.05,**P < 0.01 -
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