基于转录组测序分析美洲大蠊提取物促进小鼠创面愈合的分子机制
Regulatory Molecular Mechanism of Periplaneta americana Extracts on Wound Healing Using RNA-seq

中文摘要：美洲大蠊 Periplaneta americana是传统的中药材，其提取物已在临床上广泛用于创面治疗，但对其中的分子机制知之甚少。本文通过构建小鼠皮肤创伤模型，并基于转录组测序（RNA-seq）深入分析美洲大蠊提取物促进小鼠创面愈合的分子机制。首先构建C57小鼠皮肤全层切除模型，并将其分为3组，其中实验组分别以2种美洲大蠊提取物（康复新液、精粉）为敷料，而对照组以75%乙醇为敷料。用药处理3 d后取伤口皮肤组织送样进行RNA-seq，然后分析转录组数据找出与伤口愈合相关的差异性表达基因，并利用荧光定量PCR（Q-PCR）对伤口组织中相关基因的表达量进一步验证。实验结果显示，实验组小鼠伤口的结痂速度比对照组快，这表明了小鼠创面模型中，美洲大蠊提取物能促进伤口愈合。经转录组分析，得到显著性差异表达基因数：对照组vs.精粉组为545，对照组vs.康复新液组为938。结合生物信息分析结果和已发表的文献，发现3个可能参与调节伤口愈合的关键基因：表皮调节素（ Ereg）、 Gli-kruppel家族成员（ Gli2）和表皮型转谷氨酰胺酶 3（ Tgm3）。Q-PCR实验表明，这3个基因在美洲大蠊提取物加药组中均高表达。本文研究结果表明，2种美洲大蠊提取物：康复新液、精粉可能通过诱导 Ereg、Tgm3和 Gli2的高表达来促进皮肤创面的愈合。
英文摘要： Periplaneta americana is a traditional Chinese medicine, and its extracts have been widely used in the treatment of wound in clinic. However, the molecular mechanism is poorly understood. In this study, to investigate the regulatory mechanism of P. americana extracts on wound healing, a mouse model with excision wound was constructed to assess the efficacy of P. americana extracts on wound therapy. A total of 9 mice were randomly divided into 3 groups:control group (treated with 75% ethanol solution) and 2 P. americana extracts treated groups (Kangfuxin and Jingfen), respectively. RNA-seq was used to screen the key genes in wound skin tissues on day 3. Furthermore, real-time quantitative PCR was performed to confirm the expression of key genes. The results showed that both P. americana extracts could accelerate wound healing. RNA-seq analyses showed that, comparison to the control group, a total of 545 differentially expressed genes (DEGs) were identified in Jingfen treated group and 938 DEGs in Kangfuxin treated group. Bioinformatics analysis revealed that 3 key genes, including Ereg,Gli2 and Tgm3, may play important roles in wound healing during the treatment of P. americana extracts. Q-PCR experiments further confirmed that Ereg,Gli2 and Tgm3 were up-regulated after the treatment of both P. americana extracts. Collectively, our results indicated that the extracts of P. americana may accelerate wound healing by regulating Ereg, Gli2 and Tgm3 expression. 2017,36(4): 398-403 收稿日期：2017-03-31 DOI：10.11984/j.issn.1000-7083.20170106 分类号：Q95-33;Q786 作者简介：陈佳松(1991-),男,硕士研究生,研究方向为细胞生物学,E-mail:chenjiasong91@qq.com *通讯作者：耿福能,E-mail:haoyishenggfn@126.com;邹方东,E-mail:fundzou@scu.edu.cn 参考文献： 付小兵, 王德文. 1997. 创伤修复基础[M]. 北京:人民军医出版杜:81. 李远辉, 李希, 冯建安, 等. 2014. 美洲大蠊不同提取部位的体外抑菌活性研究[J]. 中药与临床, 5(6):27-29. 舒崇湘, 程天民, 阎国和, 等. 2001. 6Gy全身照射对皮肤伤口几种细胞外基质成分的影响及W 11-a 12的促愈作用[J]. 中华创伤杂志, 17(10):604-607. 谭巧云, 满红霞, 那凯歌, 等. 2016. 美洲大蠊提取物对口腔溃疡大鼠模型的作用[J]. 中国临床药理学杂志, 32(11):1014-1016. 伍伟明, 金丽颖, 王真权, 等.