小胶质细胞不同极化状态下P2rx7剪接变异体差异化表达  

Differential expression of P2rx7 splice variants in microglial cells under different polarization states

作　　者：安晓萌 周方宇 张霁 许学兵 AN Xiao-meng;ZHOU Fang-yu;ZHANG Ji;XU Xue-bing(Center Laboratory,Hong Kong University-Shenzhen Hospital,Shenzhen 518053,Guangdong,China;不详)

机构地区：[1]香港大学深圳医院中心实验室,广东深圳518053 [2]香港大学深圳医院临床-转化-基础科研实验室,广东深圳518053 [3]香港大学深圳医院麻醉医学部,广东深圳518053

出　　处：《广东医学》2024年第11期1413-1417,共5页Guangdong Medical Journal

基　　金：深圳市科技计划项目(JCYJ20210324114607020)。

摘　　要：目的P2X7受体(P2rx7)的选择性剪切影响P2rx7基因外显子的表达,是调节P2rx7功能的主要方式之一。本研究分析小胶质细胞M1/M2极化状态下P2rx7不同剪接异构体的差异表达。方法培养并增殖BV2永生化小鼠小胶质细胞,利用药物脂多糖(LPS,100 ng/mL)或白细胞介素-4(IL-4,40 ng/mL)分别刺激BV2细胞,培养48 h后通过流式细胞仪分析确定细胞M1/M2极化状态,并采用实时定量PCR分析小鼠P2rx7-v1、-v2、-v3、-v4和-vk等5种P2rx7剪接异构体的表达量。结果正常培养条件下小鼠小胶质细胞系BV2中所有P2rx7各转录本均可在mRNA水平检测到,其中P2rx7-v1是BV2细胞系中表达的主要形式,P2rx7-v2、-v3、-v4和-vk等异构体表达量相对较少或几乎没有。应用LPS刺激培养BV2细胞48 h后,显著抑制了M2相关标志物CD206的表达(P<0.01),M1相关标志物CD86的表达略有升高但差异无统计学意义(13.0%vs.78.8%,P=0.054),此时P2rx7-v1、-v2、-v3、-v4和-vk等异构体表达量均有明显增加(P<0.01),P2rx7-v4表达量增加相对较小(15.8%,P<0.05)。IL-4刺激培养BV2细胞48 h后,M2相关标志物CD206表达显著增加(280.2%,P<0.01),而M1相关标志物CD86表达明显减少(47.6%,P=0.035),进一步检测显示P2rx7-v1、-v4异构体表达量明显减少(64.9%vs.52.9%,P<0.01),P2rx7-v3的表达量则略有增加(61.7%,P<0.01)。结论小胶质细胞M1/M2极化状态下5种P2rx7剪接异构体呈现不同程度表达改变,进一步研究不同P2rx7剪接异构体的结构功能是否影响小胶质细胞极化状态很有必要。Objective P2X7 receptor(P2rx7)alternative splicing affects exon expression of the P2rx7 gene and is a primary regulator of P2rx7 function.This study aims to investigate the differential expression of various P2rx7 splice variants in microglial cells under M1/M2 polarization states.Methods BV2 immortalized murine microglial cells were cultured and treated with lipopolysaccharide(LPS,100 ng/mL)or interleukin-4(IL-4,40 ng/mL)for 48 hours to induce M1 or M2 polarization,respectively.Flow cytometry confirmed polarization states,and real-time quantitative PCR was employed to quantify expression levels of five P2rx7 splice variants(P2rx7-v1,-v2,-v3,-v4,and-vk).Results All P2rx7 transcripts were detectable at the mRNA level in BV2 cells under standard culture conditions,with P2rx7-v1 as the predominant variant.Other splice variants,including P2rx7-v2,-v3,-v4,and-vk,were present at relatively low levels or nearly undetectable.Following LPS treatment,expression of the M2 marker CD206 was significantly reduced(P<0.01),while the M1 marker CD86 showed a non-significant increase(13.0%vs.78.8%,P=0.054).Notably,expression levels of all P2rx7 variants increased significantly(P<0.01),except for P2rx7-v4,which exhibited a relatively modest increase(15.8%,P<0.05).In contrast,IL-4 stimulation led to a significant upregulation of CD206(280.0%,P<0.01)and a reduction in CD86(47.6%,P=0.035).Further analysis revealed decreased expression of P2rx7-v1 and-v4(64.9%vs.52.9%,P<0.01),while P2rx7-v3 expression showed a slight increase(61.7%,P<0.01).Conclusion The five P2rx7 splice variants exhibit differential expression under M1/M2 polarization states in microglial cells.These findings underscore the need for further research on the structural and functional impacts of P2rx7 splice variants on microglial polarization.

关 键 词：小胶质细胞 极化 P2X7受体 剪接变异体 

分 类 号：R614[医药卫生—麻醉学] R765[医药卫生—外科学]