微小RNA seq-14465_x69促进胎儿皮肤创伤无瘢痕愈合及其机制的研究

We have found that significantly differential expression of human novel miRNA seq-14465_x69 at different gestational ages in fetal keratinocytes (KCs). Bioinformatics analyses found seq-14465_x69 may target TGF-β2, TGF-βR2 and Smad3 which were important TGF-β/Smads pathway members. Over expression of seq-14465_x69 in human epidermic cell line HaCaT was made by transfection. The effect of seq-14465_x69 on the proliferation ability of the transfected HaCaT cells was up-regulated. Real time qPCR and Western blot have found COL-III and Has3 were highly expressed in 14465_x69-transfected HaCaT cells. After co-culture with transfected HaCaT cells, the proliferation and migration abilities of human fibroblasts (hFBs) were significantly improved. Besides, the differentiation ability of hFBs into myo-fibroblasts was significantly decreased. The results have proved that over expression of seq-14465_x69 turn the adult KCs back to the status of fetal KCs which contribute to scarless wound healing. Real time qPCR, Western blot and RNA sequencing would be used to investigate the secretion of Extracellular matrix by KCs with over expression of seq-14465_x69 and co-cultured hFBs. In vivo experiment would be used to verify the effect of KCs with over expression of seq-14465_x69 on skin wound scarless wound healing. Furthermore, Luciferase reporter experiments and Western blot were used to confirm the target genes of seq-14465_x69. The aim of the study is to provide new targets which miRNAs contribute to scarless wound healing.

人新miRNA seq-14465_x69（14465）在不同孕期胎儿角质形成细胞（KCs）中表达存在显著差异。生物信息学预测14465可能对TGF-β/Smads传导通路的重要因子TGF-β2、TGF-βR2和Smad3具有调控作用。过表达14465显著促进人表皮细胞系HaCaT的增殖能力，并显著促进细胞中COL-III和Has3的表达。与过表达14465的HaCaT细胞共培养后，真皮成纤维细胞（FBs）的增殖及迁移能力显著上调，且向肌成纤维细胞分化的能力下降。说明过表达14465可以促使成人KCs向胎儿状态转变。我们将进一步检测过表达14465的HaCaT细胞及与之共培养FBs表达细胞外基质能力的变化，并通过体内实验验证过表达14465的HaCaT细胞对皮肤创伤愈合过程中瘢痕形成的影响，验证14465的靶基因。为miRNAs调控KCs促进无瘢痕修复提供新靶点。

皮肤是人体最大的器官，与外界环境直接接触，容易受到伤害。复杂性的创面，例如烧伤，大面积创伤等情况，常常在愈合后易形成瘢痕组织。在临床上，皮肤组织再生具有广泛的需求，加快皮肤愈合速度、减轻愈合过程中瘢痕修复能够更好的帮助包括烧伤等大面积皮肤缺损患者进行创面治疗，早期帮助患者脱离困境。.孕早期至孕中期胎儿皮肤创伤后愈合速度快，并不伴有瘢痕形成及炎症反应。为了明确胎儿皮肤创伤无瘢痕愈合的机制，我们应用二代小RNA测序及实时定量PCR检测得出人微小RNA——seq-14465_x69（seq14465）在孕中期胎儿hKCs中的表达显著升高。进一步的生物信息学预测发现seq14465可能对TGF-β/Smads信号通路的重要因子TGF-β2、TGF-βR2和Smad3均具有调控作用。TGF-β/Smads信号通路的异常激活能够显著促进皮肤创面愈合过程中瘢痕的形成。我们预测孕中期胎儿hKCs中高表达的seq14465可能通过调控TGF-β/Smads信号通路促进皮肤组织再生。.为了研究seq14465对皮肤再生能力的影响，本研究通过体外实验及体内实验证明，seq14465能够通过促进HaCaT细胞增殖能力及hFBs的迁移能力促进皮肤创面愈合速度、缩短愈合时间。并进一步通过测序、实时定量PCR及Western blot证明seq14465通过调控瘢痕形成相关蛋白TGF-β2、COL-I及COL-III的表达水平减少皮肤创面愈合后瘢痕组织的面积，并且能够促进创缘处皮肤及皮肤附属器的组织形态发育，使皮下胶原排列更加整齐。.为了探讨seq14465促进皮肤再生的机制，我们通过双荧光素酶报告基因检测分析及Western Blot验证seq14465对TGF-β/Smads信号通路相关基因TGF-β2、TGF-βR2和Smad3具有靶向调控作用。实验证明seq14465通过抑制TGF-β/Smads信号通路相关基因促进皮肤创面处组织再生。.微小RNA是一类作用广泛的分子，目前，有国家已经具备将微小RNA应用于临床治疗的技术，微小RNA药物的研发正处于关键阶段，需要对其进行系统全面的分析以达到治疗效果。本研究通过大量实验证明了seq14465能够通过抑制TGF-β/Smads信号通路相关基因促进皮肤创面处组织再生。对未来用小RNA临床治疗难治性皮肤创面有重要意义。