基于CNC生物网络图及在体、离体实验差析养阴、回阳生肌机制

The lncRNA becomes a research hotspot due to that it has been found to play an important role in regulating biological functions of gene transcription in recent years and the CNC co-expression network proves to be a foundation for selection of molecular targets of many diseases and candidate genes.The author found the exploration and discussion of the method Yang Yin Sheng Ji and Hui Yang Sheng Ji was so little in IncRNA aspect after gathering and analysis of traditional Chinese medicine external Sheng Ji treatment literature in nearly.six years.Combined with the production of the previous studies of Yang yin ShengJi method,this experiment will adopt Go、subgroup 、KEGG Pathway analysis and construct CNC Co-expression network diagram for the target IncRNA to clear the IncRNA of affecting the wound healing and the distinction of regulation betweenYang Yin and Hui Yang method by microarray chips screen lncRNA for group of acute wound and the group of chronic impaired healing wound 、the group Yang Yin Sheng Ji and the group Hui Yang Sheng Ji to select chips.The vivo experiment will detect the dynamic curve of different time points of 8 target lncRNAs in the wound of mice by Real-time PCR.The vitro experiment will apply fluorescence microscopy and flow cytometry instrument, Brdu staining and Scratch Assay to observe the biological characteristics of keratinocyte after planning to build the target lncRNA plasmid and make it silent or expression.All that can verify the function of the IncRNA in wound healing and compare and analyse the regulation mechanism of the two methods of Yang Yin Sheng Ji and Hui Yang Sheng Ji.

近年来lncRNA被发现在基因转录的生物功能中发挥重要的调控作用而成为研究热点，CNC网络图成为许多疾病筛选分子靶点和候选基因的依据，笔者荟萃近6年中医外治生肌文献，发现养阴与回阳法从lncRNA探讨颇少，结合前期对养阴生肌做出的研究，本实验应用微阵列芯片对临床样本急、慢性难愈合创面、养阴生肌组与回阳生肌组进行lncRNA芯片筛选，对目标mRNA进行 GO、 亚组、 KEGG Pathway分析、搭建CNC网络图，明晰影响创面难愈合的lncRNA及养阴与回阳调控差异；在体实验利用Real-time PCR 检测8个目标lncRNA在小鼠各创面不同时间点的动态曲线；离体实验拟构建目标lncRNA质粒，沉默或过表达后应用荧光显微技术、流式细胞仪、Brdu 染色及Scratch Assay 观察角质形成细胞生物学情况，验证lncRNA在创面修复功能及差析养阴回阳生肌调控机制。

中医药外治促愈具有简便廉验的优点，但诸多机制不明，特别在LncRNA研究颇少，本课题在前期基础利用高通量测序观察急、慢创面、养阴、回阳组创面基因表达图，GO、KEGG分析、pathwany展示，得到急慢创组LncRNA、mRNAs有差异，KEGG分析破骨细胞通路上调、细胞因子通路受体下调，Pathway Map展示筛选出IL6、IL18等8个指标，5个通过验证，搭建CNC图，发现20个LncRNA正、负调控IL6或IL18来影响愈合。养阴组有2242有差异，ENST00000443364、576797、620167通过验证，GO分析发现养阴药物顺、反式调控表皮细胞影响巨噬细胞促进炎性因子分泌、转移，并调控VSMC和EC的增殖和迁移，KEGG 分析IBD、MAPK、AMPK通路与创面修复相关。回阳组有1792个差异，ENST00000450589通过验证，GO分析发现LncRNA-cis功能富集在角质化、皮肤发育、角蛋白、表皮分化、细胞骨架的结构成分，LncRNA-trans功能富集在阴离子运输、细胞骨架蛋白结合、神经元部分等部分，KEGG分析T细胞受体信号、内吞作用、细胞凋亡、花生四烯酸通路影响促愈，结合韦恩图发现ENST00000450589与细胞死亡、血管生成、免疫稳态、造血、抗凝、抗炎相关，靶标基因为KLHL20、DARS2等6个；ENST00000596066与促愈相关，靶标为IGFL3；ENST00000652702与EGF、血管生成、皮肤修复相关，靶标为FGFR2、TGM5。动物实验验证lncRNA H19在高糖下表达下降，养阴、回阳组H19上升，后期回阳组H19逐渐下降、养阴组不断上升，证明养阴生肌促进胶原蛋白及肉芽形成、减少瘢痕形成等，回阳生肌方有促进细胞增殖、胶原蛋白及消炎等作用；细胞实验通过CCK-8、划痕实验、ELISA等发现养阴药调控H19早期促进细胞凋亡，加速组织脱落，后期促进HaCaT细胞增殖、迁移，再上皮化，避免瘢痕形成，同时调控TNF-a/IL-6的表达，减缓对HaCaT的的刺激，但回阳组在细胞实验中没有明显作用，本实验明确了急、慢、养阴、回阳组各创面lncRNA-mRNA差异，分析了功能富集及位点关系、通路及靶标机制差异，揭示了养阴与回阳调控lncRNA在创面修复的生物角色及表达特征，为中医外治促愈开创了新思路，提供了科学依据。