生肌玉红胶原诱导脂肪干细胞启动miRNA-LncRNA靶向调控P38、P53路径促进血管新生与减少瘢痕愈合效应机制研究

Based on the findings of improving angiogenesis and reducing scar formation during healing process with regulations of protein pathways of AKT and TGF-βinside adipose derived stem cells with a kind of typical Chinese traditional topical biomaterial named as Shengji Yuhong collagen application from the former national natural science grant and the primary results of positive regulated targeted miRNAs and LncRNAs related with gene pathway of P38 and P53 of adipose derived stem cells treated with Shengji Yuhong collagen through high-throughput genome sequencing, the project will furtherly screen the most selected different miRNA and LncRNA targeted to the pathway of P38 and P53 by way of q-PCR and fixed-point gene mutation assays regulated by Shengji Yuhong collagen, followed by the determination of selected targeted genes which conjugated with the aimed proteins through methods of immune coprecipitation as well as authenticate reverse with the selected targeted genes silence, mutation and/or blank mimic assays. Ultimately, in vivo experiments will be adopted to confirm the mechanisms of improving wound healing and reducing scar formation of adipose derived stem cells regulated by Shengji Yuhong collagen as well as the interactions with surrounding tissues..The gene mechanism on wound healing of Shengji Yuhong collagen regulated adipose stem cells would be explored in the proposal project, which lays the necessary basis of put forward a kind of novel biomaterial ascribed to TCM of self-intellectual property and clear target points as well as optimal controlled compositions with the functions of fast angiogenesis and improving wound healing benefits to clinical transformation and international application in the future researches.

在前期国家自然基金发现生肌玉红胶原显著调控脂肪干细胞AKT及TGF-β蛋白通路以促进血管新生及减少瘢痕愈合基础上，基于预实验高通量基因测序筛选出生肌玉红膏调控P38、P53通路差异显著靶向长链非编码（LncRNA）及小分子RNA（miRNA），本研究进一步通过RT-PCR法及定点基因突变法确定生肌玉红胶原调控差异最显著之靶向RNA，采用免疫共沉淀法确定差异最显著的RNA靶向基因与目标蛋白结合机制并反向验证，建立裸鼠后肢缺血模型，以靶向报告基因免疫荧光及空白类似物转染测定，确定生肌玉红胶原调控脂肪干细胞促进愈合与减轻瘢痕在体作用机制及与周围组织交互关系。.本申报在基因层面上探索生肌玉红胶原调控脂肪干细胞功能，形成高新且具有自主知识产权、作用靶点明确、成分优化合理可控的新型快速血管化以促进组织修复之中医药生物材料，为今后中医新型材料国际化并临床转化奠定必要的基础。

在前期国家自然基金发现生肌玉红胶原显著调控脂肪干细胞AKT及TGF-β蛋白通路以促进血管新生及减少瘢痕愈合基础上，通过细胞与裸鼠实验，采用RT-qPCR、慢病毒转染静默基因、荧光GFP表达、免疫共沉淀及血管斑点成像、病理免疫组化等实验技术，证明了生肌玉红胶原通过通过旁分泌外泌体携带调控因子miR-21调控lncRNA TCONS_00178845从而影响miR-21/ lncRNA TCONS_00178845/p38信号调节通路发挥促血管新生作用，在创伤修复期达到促进愈合的目的。在裸鼠下肢缺血实验中可以达到缺血后肢血供完全恢复的目标；在创伤愈合塑型期，生肌玉红胶原启动miR203靶向调控P53可以显著减少瘢痕愈合，提高愈合质量。结果显示生肌玉红胶原在创伤相关修复期对目标基因miR-21、TCONS_00178845及miR203调节效果相对对照组达到1.5至2.5倍，对相关蛋白及功能基因：AKT/ERK及P38、P53的表达为对照组的3倍以上。均取得了显著性统计学差异。.得出重要结论如下：1. 胶原酶消化离心脂肪组织法可以获得纯度较高的脂肪干细胞，可以保证足够的细胞数量、细胞干性、较强的分化能力。2. 后肢股动脉结扎造模方法，可以维持较长时间的缺血状态，适用于裸鼠动物模型。3. 生肌玉红胶原可以有效地调控脂肪干细胞促进血管新生，且对肝、心、脾、肺、肾无明显毒性。4. 生肌玉红胶原能通过促进hADSCs VEGF的释放，通过AKT/ERK途径，促进血管新生，机制通过miR-21/ lncRNA TCONS_00178845/p38的调节通路来调控脂肪干细胞功能；通过与瘢痕及成纤维细胞培养，通过调节miR203/P53减轻愈合瘢痕形成。5. 生肌玉红胶原可能通过干预脂肪干细胞分化，参与血管新生与胶原排列改善愈合并较少瘢痕形成。.本研究首次发现了对经典外用药生肌玉红膏进行了制剂改良优化，使生肌玉红膏与可降解胶原完全结合，形成可吸收降解的生肌玉红胶原，获得国家发明专利；运用范围可以植入体内，促进组织再生，对心血管、下肢缺血性疾病及软组织填充整形方面可能提供新的治疗手段，扩大中医外用制剂运用范围，同时探索出其对脂肪干细胞的调控机制与通路，为后期开发转化奠定了一定的基础。本研究培养4名中医博士研究生，培养出可持续研究团队。

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