重组发形霞水母多肽生长因子Cc-GRN-1的生物学活性与作用机制

Large biomass of jellyfish is one of the most important resources in marine ecosystems. It is reported that jellyfish has various biological characteristics, including strong reproductive capacity, fast growth and strong regeneration ability. In our previous study, we have isolated a granulin-like polypeptide growth factor (Cc-GRN-1), with a molecular weight of 5782.9 Da, from the tentacle extract of Cyanea capillata (C. capillata). And the full-length sequence of Cc-GRN-1 contains 58 amino acids and six disulfide bonds. Thus, this study aims to recombinantly express this active polypeptide by molecular cloning and to explore its biological activities. Firstly, cell damage, repair, migration and pro-angiogenesis experiments would be conducted to comprehensively analyze the growth-promoting effects of Cc-GRN-1 at cell and tissue levels, and the effects of Cc-GRN-1 on cell cycle progression would be also analyzed using flow cytometry. Secondly, the phosphorylation levels of some proteins, including NF-κB, Akt, ERK1/2 and JNK, and cell cycle related proteins, including CyclinB1 and CyclinD1, would be detected to clarify the mechanism of its survival and apoptotic-related signaling pathways. Finally, an animal model of seawater immersion injury would be established and used to compare the wound-healing effects between recombinant Cc-GRN-1 and recombinant human epidermal growth factor. Through this study, it is expected to provide a new idea to solve the problem of wound healing in the injuries combined with seawater immersion.

水母生物总量庞大，是一类重要的海洋生物资源。我们注意到水母繁殖能力强，生长发育快，同时具有很强的再生修复能力，前期从发形霞水母触手组织定向分离得到一个分子量为5782.9 Da的颗粒蛋白样多肽生长因子Cc-GRN-1，经测定其全长序列由58个氨基酸组成，共有6个二硫键。本课题拟通过分子克隆重组表达该活性多肽；通过细胞损伤修复、细胞迁移和促血管新生等实验，从细胞、组织水平综合分析Cc-GRN-1的促生长效应，并利用流式细胞仪分析Cc-GRN-1对细胞周期进程的影响；检测NF-κB、Akt、ERK1/2、JNK等蛋白磷酸化水平以及细胞周期相关蛋白CyclinB1、CyclinD1表达水平，阐明其对生长和凋亡相关信号通路的作用机制；进一步制作海水浸泡伤动物模型，对比分析重组Cc-GRN-1和重组人表皮生长因子对海水浸泡伤创面的愈合促进效果。可望为解决海水浸泡伤创面愈合这一难题提供新的思路。

水母生物总量庞大，是一类重要的海洋生物资源。已知水母繁殖能力强，生长发育快，其触手具有很强的再生修复能力。本项目首先利用CCK-8法确定发形霞水母（Cyanea capillata）触手提取物（tentacle extract，TE）在一定剂量范围(0.5~2 μg/ml)可以促进人脐静脉内皮细胞（HUVECs）生长增殖，然后组合应用超滤、Superdex 30 prep grad、MZ-ANALYTICAL C8和Vydac C18色谱柱等多步纯化，从TE中分离获得了分子量为5782.9 Da的多肽生长因子CcGRN-1。接下来的生物学效应分析中，首先观察了CcGRN-1促细胞增殖作用的时间-剂量-效应关系，发现CcGRN-1对HUVECs具有剂量和时间依赖性的促增殖作用；然后利用流式细胞仪和Western blotting检测，发现CcGRN-1可以诱导HUVECs从G1期进展至S/G2期，并明显上调细胞周期蛋白CyclinB1和CyclinD1的表达，表明CcGRN-1可以加快HUVECs细胞周期进程；还通过细胞划痕试验和Transwell细胞体外侵袭实验发现，CcGRN-1可以显著诱导HUVECs迁移和侵袭，且CcGRN-1促细胞迁移和侵袭效应呈时间依赖性。明确CcGRN-1的促细胞生长增殖效应后，我们进一步分析了CcGRN-1对相关信号通路的影响，结果表明CcGRN-1激活PI3K/Akt、ERK1/2 MAPK信号通路，但不影响JNK MAPK和NF-κB信号通路，其中ERK1/2 MAPK（而非PI3K/Akt）信号通路与CcGRN-1促进细胞周期进展相关。综合以上结果可以推测，CcGRN-1诱导的细胞增殖和迁移主要与ERK1/2 MAPK信号通路相关。