促皮肤创面修复和虫体断肢再生的美洲大蠊活性多肽研究

Cockroaches, including the American cockroach Periplaneta Americana, are known as “little toughness” with extremely strong capacity of wound healing and amputation regeneration. The Chinese medicine named “rehabilitated new liquid” is made of dry extracts from P. Americana and clinically used for healing incised wounds and burns. The repair process in both insect and human could involve cell division and differentiation promoted by active polypeptides. However, the active ingredients and their functional mechanism as well as the internal link from cockroach to human are nearly unknown. Our lab has sequenced the whole genome from P. Americana, and verified its ability of wound healing and limb regeneration. This project will be focused on the following items: (1) To identify the active polypeptides in vitro with the injured human skin cell lines and skin stem cells for bioassay by combing HPLC separation, mass spectrometry assay, and genome blast, and further confirm their roles in vivo by using the skin-injured mouse model. (2) To observe if RNAi knockdown of the polypeptide genes could decrease wound healing and limb regeneration in the cockroach. (3) To assess if the polypeptides activate cell division and differentiation via the JNK, Grainy head, Wnt, Notch, TGF-beta, and Hedgehog signal transduction pathways in both mammals and cockroach. This study will help unravel the life mystery of cockroach and drive the development of traditional Chinese medicine.

美洲大蠊（俗称蟑螂）素有“小强”之称，有极强的伤口愈合和断肢再生能力；干燥虫体提取物制备的中成药“康复新液”含活性多肽有效成分，可促进创面修复，治疗金创和烧烫伤具有良效。两者均涉及到活性多肽促进细胞分裂、迁移和分化，但其多肽成分、作用机制、内在联系均不清楚。申请人实验室已测定了美洲大蠊的基因组，验证了其伤口愈合和断肢再生能力。本项目拟（1）以人皮肤细胞系和表皮干细胞为生测对象，结合HPLC分离、质谱分析和基因组比对鉴定活性多肽，并在小鼠创面修复模型中验证；（2）RNA干扰该活性多肽编码基因观察是否降低美洲大蠊伤口愈合和断肢再生的能力；（3）在哺乳动物和美洲大蠊中确定该活性多肽是否通过JNK, Grainy head, Wnt, Notch, TGF-beta和 Hedgehog 等信号通路诱导细胞分裂和分化。研究成果不仅有助于揭示“小强”的生命奥秘，还对我国传统昆虫中药具有弘扬和推动作用。

组织再生和伤口修复是医学研究的热点和难点。通过水提和醇提方法提取美洲大蠊，采用MTT细胞增殖实验检测其对小鼠成纤维细胞增殖的影响；通过MSF细胞划痕模型检测对小鼠成纤维细胞迁移的影响；通过小鼠皮肤损伤模型探究对小鼠创口修复的影响；最后用转录组学分析了哪些信号通路参与了组织再生与修复。结果显示两种美洲大蠊的提取物在体外能促进小鼠成纤维细胞的增殖。醇提比水提有较好的促进细胞增殖的作用，细胞存活率也会增加。划痕实验证明两种提取方法在体外能促进小鼠成纤维细胞的迁移。小鼠创口损伤实验结果可知: 美洲大蠊的水提物和醇提物均可促进MSF细胞增殖与迁移，加速小鼠创面皮肤愈合，美洲大蠊醇提物促进创口修复的作用效果显著，且用90%乙醇效果最好。转录组进一步揭示参与损伤修复的信号通路是hippo,TGF-beta 和Hedgehog，每个通路具体基因功能需要进一步深入研究。