HMG(High-Mobility Group Box Protein)在刺参(Apostichopus japonicus)肠道再生期间的表达情况分析

Expression Analysis of HMG (High-Mobility Group Box Protein) During the Intestine Regeneration of Sea Cucumber Apostichopus japonicus

关键词:  刺参  HMG  肠道再生  qRT-PCR  免疫荧光

Key words:  Apostichopus japonicus  HMG  Intestine regeneration  qRT-PCR  Immunofluorescence

摘要:

运用实时荧光定量PCR (qRT-PCR)技术，测定了一种核酸内非组蛋白类蛋白HMG(High-mobility group box protein)在刺参肠道再生第30分钟、1、2、6小时、1、3、7天的mRNA表达情况，结果显示，再生第6小时之前，表达量与正常刺参肠道相比无显著变化，再生第1–7天，其表达量显著高于对照组，并于第7天达到最大值。采用免疫荧光技术，分析测定了HMG在刺参肠道再生各时期的蛋白表达情况，结果显示，再生第2、6小时，HMG蛋白荧光信号弱，仅在浆膜层和黏膜层有少量分布；再生第1–7天，HMG蛋白荧光信号逐渐增强，再生第1天的HMG荧光主要分布在浆膜层和肌肉层，再生第3天和第7天的荧光信号最强，各层均有分布。结合HMG在其他物种组织修复再生中的作用，推测HMG可使免疫系统识别损伤信号，启动修复再生功能，调控细胞外基质(ECM)成分的降解与生成，诱导干细胞迁移、分化，并可能在伤口愈合阶段过后参与细胞免疫。

Abstract:

High-mobility group box protein (HMG) is a novel cytokine that takes part in inflammation, cell migration, and tissue damage and regeneration. Regeneration usually involves the regrowth of cells, tissues and organs, and plays an essential role in the survival and adaptation of a species. Sea cucumbers Apostichopus japonicus (Echinodermata, Holothuroidea), which are phylogenetically related to invertebrates, are capable of regenerating damaged organs and body parts, such as the intestine, the respiratory tree, the gonads and the body wall. Sea cucumbers can eviscerate intestines and other organs through the cloaca in response to external stimulations. Within a few weeks after losing an organ or an appendage, the sea cucumber is able to regenerate the missing parts. These animals are thus excellent candidate models for research on organ regeneration. In this study, we analyzed the expression of mRNA and proteins of HMG during the intestine regeneration in sea cucumber using qRT-PCR and immunofluorescence techniques, respectively. The results showed that compared to the control group, there was no significant rise in the expression of either mRNA or proteins of HMG from 30-minute to 6-hour post-evisceration. HMG mRNA expression was significantly upregulated from 1-day to 7-day post-evisceration. The protein expression was higher as well compared to the control group. These results suggested that HMG was involved in the intestine regeneration of A. japonicus and could participate in the activation of the innate immune system in response to tissue damages, and hence was required in the processes such as repair, remolding of extracellular matrix (ECM), and the induction of stem cell migration and differentiation.