致痛性操作对新生大鼠远期海马基因表达谱的影响

The effects of painful procedures during neonatal period on rats'hippocampal gene expression profiles in later life

目的探讨致痛性操作对新生大鼠远期海马基因表达谱的影响.方法将28只新生雄鼠随机分为疼痛组和对照组,每组14只.疼痛组大鼠生后第1~7天每日进行4次针刺足底的致痛性操作,建立新生期致痛经历的大鼠模型,模拟新生儿重症监护病房反复经历致痛性操作的患儿;对照组用棉签触碰同侧足底.分别取第8天和第21天两组大鼠的海马组织进行转录组测序,构建基因表达谱并筛选出相关差异基因进行功能分析.采用实时荧光定量聚合酶链反应对与疼痛刺激反应或大脑认知相关的差异表达基因进行验证,包括γ-氨基丁酸A型受体β1亚基编码基因(GABRB1)、谷氨酸NMDA受体NR2A亚基编码基因(GRIN2A)和白细胞介素1受体相关蛋白编码基因(IL1 RAPL1),蛋白免疫印迹法验证其中1个关键基因.结果测序结果显示,疼痛组和对照组第8天的差异基因仅有6个,而第21天增加至53个,其中下调者占85.0%(45/53),根据基因本体功能和京都基因与基因组百科全书注释结果,差异基因功能主要富集在细胞膜、电压门控离子通道、突触连接、神经递质受体、免疫反应等.关键基因的实时荧光定量聚合酶链反应和蛋白免疫印迹验证结果与转录组全基因测序RNA序列趋势一致.结论早期反复经历致痛性操作的大鼠大脑海马突触细胞膜上的电压门控离子通道蛋白、神经递质受体以及一些关键基因如GABRB1等差异表达,初步阐释了早期疼痛影响新生儿脑发育可能的分子机制.

Objective To study the effects of painful procedures during neonatal period on rats'hippocampal gene expression in later life.Method A total of 28 newborn male rats were randomly assigned into two equal groups.From day1(P1)to day7(P7)after birth,rats in the pain group received four times of needle acupuncture at the foot everyday,simulating the infants'experience in the NICU.Rats in control group were touched at the same side of the foot with a cotton swab.Transcriptome sequencing of the hippocampus of the two groups were examined on day 8(P8)and day 21(P21).The gene expression profiles were established and the differentially expressed genes were screened for functional analysis.Quantitative Real-time polymerase chain reaction(qRT-PCR)was used to confirm these differentially expressed genes(GABRB1,GRIN2A,IL1 RAPL1)related to pain-stimulated response or brain cognition,and one of the key genes was further verified using Western blotting.Result The sequencing results showed that there were only 6 differentially expressed genes in hippocampal tissues of rats on P8 in the two groups.Howerer,the number increased to 53 on P21 and 85.0%of these genes were down-regulated(45/53).Gene Ontology(GO)function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis showed that the differentially expressed genes were mainly expressed on the cell membrane,voltage-gated ion channels,synapses,neurotransmitter receptors,immune responses,etc.The qRT-PCR and Western-blot results of key genes were consistent with the transcriptome sequencing.Conclusion Pain stimuli at an early stage after birth may trigger differentially expression of voltage-gated ion channel proteins,neurotransmitter receptors,and some key genes such as GABRB1 on hippocampal synaptic cell membranes in rats.These phenomenon may provide initial explanation for the molecular mechanism of early pain stimuli on neonatal brain development.