诱导山羊表皮干细胞向角膜上皮细胞分化相关miRNAs的功能研究

Exploring the molecular mechanism of inducing epidermal stem cells into corneal epithelial cell is a hot point in the stem cell researhing field .Emerging evidence suggests that microRNA(miRNAs),an abundant class of 21～22nucleotide small regulatory RNAs, play key role in controlling the post-transcriptional genetic programs in stem and progenitor cells, however there is little evidence show how miRNAs regulating the transition from stem cells to another type of cells.To explore miRNA expression change of differention of goat epidermal stem cell (EpiSCs) into Corneal epithelial cells(CECs),in this study goat EpiSCs were induced into CECs to compare the genetic patterns between EpiSCs and CECs by a large genomewide expression profiling of mRNAs and miRNAs. EpiSCs and induced CECs were collected,total RNA was extracted and purified. After fluorescent tags being added, hybridization was carried out on microRNA chip. SAM analysis was performed to find out the significant expression difference,then the difference verified by quantitative RT-PCR, MiRBase and target gene analysis were employed for the prediction of miRNA targets. To explore how some specific miRNAs regulating the transition from epidermal stem cell to corneal epithelial cell through overexpression, or inhibiton of those specific miRNAs, and identify this process by luciferase repoter assays, finally the miRNAs function were predicted by GO analysis.The putative findings and experimental approach will provide a suitable system to directly interrogate the role of miRNAs during corneal epithelium differentiation of epidermal ste m cells.

探索表皮干细胞转分化为角膜上皮细胞的分子机制是目前干细胞转分化研究领域的热点之一。微RNA（microRNAs,miRNAs）是生物体内最重要的基因调控因子，前期研究已证明并miRNAs调控干细胞增殖分化过程，但miRNAs如何调控干细胞转分化报道甚少。因此，本项目以山羊表皮干细胞为研究对象，拟采用miRNA芯片和mRNA芯片构建诱导山羊表皮干细胞向角膜上皮细胞转分化不同阶段的miRNAs和mRNA表达谱，筛选差异表达miRNAs，对有显著差异的miRNAs进行实时定量PCR验证，并预测和验证其靶基因；通过抑制表达或过表达技术研究特定miRNAs对表皮干细胞向角膜上皮细胞转分化的影响，双荧光素酶报告基因系统验证特定miRNAs对靶基因的调控作用，对靶基因进行GO功能注释，从转录后调控水平探索表皮干细胞转分化为角膜上皮细胞的分子机制，为明晰干细胞转分化机制提供科学依据，具有重要理论意义。

目前关于miRNAs调控表皮干细胞向角膜上皮细胞转分化分子机制的研究报道甚少，本项目用transwell共培养系统诱导山羊表皮干细胞向角膜上皮细胞分化，免疫组织化学方法对被诱导细胞进行鉴定；收集山羊表皮干细胞、诱导分化细胞、角膜上皮细胞进行基因表达及miRNAs测序分析，主要结果如下：（1）用无血清、无饲养层培养体系分别建立了山羊皮肤表皮干细胞和角膜上皮细胞系，并冷冻保存20x107的干细胞。（2）用免疫组织化学方法对所分离的表皮干细胞进行鉴定，结果显示所建立的细胞系表达P63、 CK19等表皮干细胞特异性标志，具有表皮干细胞特征。（3）用transwell共培养系统成功诱导山羊表皮干细胞转分化角膜上皮细胞，并用免疫组织化学方法进行了鉴定，结果表明角膜基质细胞分泌的细胞因子可诱导皮肤表皮干细胞向角膜上皮细胞分化，为深入研究干细胞转分化机理打下了基础。（4）收集山羊表皮干细胞、诱导分化细胞、角膜上皮细胞送上海伯豪生物技术有限公司(http://www.shbiotech.org)进行基因表达谱分析（单色标记芯片实验样品杂交及分析实验合同编号：BH13289）和miRNAs测序分析（二代测序实验同编号：BC13122）工作正在进行，预期结果将筛选出与诱导表皮干细胞转分化为角膜上皮细胞过程特异相关的miRNAs，并预测其靶基因及信号通路，为下一步深入研究miRNAs调控表皮干细胞转分化的分子机制打下坚实基础。