髓样细胞触发受体2在酪氨酸激酶结合蛋白基因敲除小鼠不同脑区的表达

Expressions of triggering receptor expressed on myeloid cell 2 in different brain regions in tyrosine kinase binding protein gene knockout mice

目的比较髓样细胞触发受体2(triggering receptor expressed on myeloid cell 2,TREM2)在不同月龄酪氨酸激酶结合蛋白(tyrosine kinase binding protein,TYROBP)基因敲除小鼠和野生型小鼠不同脑区的表达差异,探讨TREM2、TYROBP与早发型阿尔茨海默病(early onset Alzheimer's disease,EOAD)的关系。方法健康TYROBP基因敲除的小鼠根据基因测序结果分成3组:纯合型(TYROBP-/-)组、杂合型(TYROBP-/+)组和野生型(wild type,WT)组,3组小鼠分别在2、4、6月龄各选10只,运用Western blot和RT-qPCR技术检测TREM2在前额叶、海马中的表达情况。结果(1)在前额叶中:Western blot和RT-qPCR共同显示,与各月龄WT组[2月龄:(0.993±0.048),(1.654±0.033);4月龄:(0.503±0.019),(2.169±0.023);6月龄:(0.600±0.036),(1.468±0.057)]相比,TREM2蛋白和mRNA在2月龄TYROBP-/+组[(0.746±0.062),(1.137±0.067)]和TYROBP-/-组[(0.661±0.028),(0.644±0.012)]的表达均降低,而在4月龄和6月龄TYROBP-/+组[4月龄:(1.140±0.006),(5.483±0.088);6月龄:(0.827±0.043),(3.020±0.082)]和TYROBP-/-组[4月龄:(1.071±0.010),(3.012±0.150);6月龄:(0.627±0.026),(1.633±0.027)]的表达均升高,差异具有统计学意义(F=12.946,134.445;725.318,289.202;12.172,202.791;均P<0.05),且4月龄小鼠升高更明显。(2)在海马中:Western blot显示,与各月龄WT组[2月龄:(1.268±0.036);4月龄:(0.813±0.010);6月龄:(0.312±0.021)]相比,TREM2蛋白在2月龄TYROBP-/+组[(0.804±0.034)]和TYROBP-/-组[(0.534±0.020)]的表达降低,而在4月龄和6月龄TYROBP-/+组[(0.932±0.011);(0.769±0.031)]和TYROBP-/-组[(0.910±0.014);(0.609±0.018)]的表达均升高,差异具有统计学意义(F=142.807,27.884,94.067;均P<0.05),且4月龄小鼠升高更明显。结论TREM2在2月龄TYROBP基因敲除小鼠脑组织中表达降低,在4月龄和6月龄TYROBP基因敲除小鼠脑组织中表达升高,推测TREM2/TYROBP信号通路参与EOAD的病理过程并且在EOAD的不同病理阶段发挥着不同的作用。

Objective To compare the expression of myeloid cell trigger receptor expressed on myoid cell 2(TREM2)in different brain regions of tyrosine kinase binding protein(TYROBP)knockout mice and wild-type mice at different months of age,and to explore the relationship between TREM2,TYROBP and early onset Alzheimer's disease(EOAD).Methods Healthy TYROBP gene knockout mice were divided into three groups according to the results of gene sequencing:the homozygous(TYROBP-/-)group,the heterozygous(TYROBP-/+)group,and the wild type(WT)group.Western blot and RT-qPCR were used to detect the expression of TREM2 in prefrontal cortex and hippocampus of 2,4 and 6 month old mice in the three groups and with 10 in each group at each time point.Results(1)In the prefrontal cortex:Western blot and RT-qPCR results showed that compared with WT mice(2-month-old:(0.993±0.048),(1.654±0.033);4-month-old:(0.503±0.019),(2.169±0.023);6-month-old:(0.600±0.036),(1.468±0.057)),the levels of TREM2 protein and mRNA in 2-month-old TYROBP-/+group((0.746±0.062),(1.137±0.067))and TYROBP-/-group((0.661±0.028),(0.644±0.012))were decreased.While in 4-month-old and 6-month-old TYROBP-/+group(4-month-old:(1.140±0.006),(5.483±0.088);6-month-old:(0.827±0.043),(3.020±0.082))and TYROBP-/-group(4-month-old:(1.071±0.010),(3.012±0.150);6-month-old:(0.627±0.026),(1.633±0.027))were increased,especially in 4-month-old mice and the differences were statistically significant(F=12.946,134.445;725.318,289.202;12.172,202.791;all P<0.05).(2)In the hippocampus:Western blot results showed that compared with WT mice(2-month-old:(1.268±0.036);4-month-old:(0.813±0.010);6-month-old:(0.312±0.021)),the level of TREM2 protein in 2-month-old TYROBP-/+group((0.804±0.034))and TYROBP-/-group((0.534±0.020))were decreased.While in 4-month-old and 6-month-old TYROBP-/+group((0.932±0.011);(0.769±0.031))and TYROBP-/-group((0.910±0.014);(0.609±0.018))were increased,especially in 4-month-old mice and the differences were statistically significant(F=142.807;27.884;94.067;all P<0.05).Conclusion The expression level of TREM2 decreases in 2-month-old TYROBP gene knockout mice while increases in 4-month-old and 6-month-old TYROBP gene knockout mice.It is presumed that TREM2/TYROBP signal pathway participates in the pathological process of EOAD and plays different roles in different pathological stages of EOAD.