miR-150 调控Notch3 对脂肪干细胞成脂分化的影响

MicroRNAs (miRNAs) are endogenous, highly conserved, short noncoding 22 nucleotide RNAs, and constitute a novel class of gene expression regulators which play important roles in various aspects of development, homeostasis, and disease. Recent studies have suggested miRNAs play an important role in stem cell self-renewal and differentiation. Our previous study found that miR-150 was expressed in the adipose stem cells (ADSCs).The preliminary data showed that miR-150 was closely associated with adipogenic differentiation of ADSCs. The mechanism may be that miR-150 targets Notch3, then decreases Notch3 expression. The decreased Notch3 will promote adipogenic differentiation of ADSCs. Based on our previous work, the project will be focused on: (1) Overexpression or silence miR-150 expression in the ADSCs, to observe its effect on the adipogenic process in vitro and in the athymic mice. (2) Investigate the regulation effect of miR-150 on Notch3 in the ADSCs. Apply the fluorescent reporter gene system to verify the direct effect of miR-150 on Notch3 and to search the binding sites with Notch3..(3) To verify the role of Notch3 in adipogenic process of ADSCs, and to explorethe new signaling pathways on Notch3 effect on adipogenic differentiation of ADSCs. (4) To verify that miR-150 regulate adipogenic gene expression during adipocyte differentiation from ADSCs by repressing Notch3. (5)Constructing the tissue engineering adiose tissue by 3D biological printing method, to further explore the miR-150 effect on the adipogenic differention in ADSCs. This study can not only reveal the adipogenic differentiation mechanism of miR-150 targeting notch3 in ADSCs, but also provide a new approach and theoretical basis for the use of adipose stem cells to treat soft tissue injuries in different body locations.

miRNA在调控干细胞自我更新和分化过程中发挥重要作用。申请者前期发现脂肪干细胞（ADSCs）表达的miR-150具有促进ADSCs向成脂方向分化的作用。其机制可能是miR-150通过作用Notch3,下调Notch3的表达，从而促进ADSCs成脂分化。本项目拟在前期工作基础上:(1)过表达或沉默miR-150在ADSCs中的表达，进行成脂诱导，在体外及裸鼠体内观察其对成脂过程的影响。(2)观察miR-150对Notch3的调节，验证miR-150与Notch3的直接作用和结合位点。(3)验证Notch3在ADSCs成脂过程中的作用。(4)验证miR-150通过作用Notch3促进ADSCs成脂分化的机制。(5)应用3D生物打印技术将过表达miR-150的ADSCs与脂肪脱细胞外基质打印，构建组织工程脂肪。通过本课题的研究，将揭示miR-150在ADSCs分化中的作用机制。

脂肪组织工程是修复软组织缺损有效的治疗方法。MicroRNA（miRNA）影响干细胞许多功能，包括干细胞的动员，增殖和分化。miR-150在脂肪干细胞（ADSCs）中的作用尚不清楚。在本项研究中，我们研究了miR-150对ADSCs增殖、成脂和成骨分化的影响。从野生型（WT）和miR-150基因敲除（KO）小鼠腹股沟脂肪组织中分离ADSC，流式细胞术鉴定。通过RT-PCR证实miR-150在ADSC表达。通过miR-150或Notch3的小干扰RNA（siRNA）转染ADSC。在miR-150敲低和对照ADSC中进行MTT分析和集落形成分析。与野生型小鼠相比，miR-150基因敲低显着降低了ADSCs成脂分化的能力。miR-150的过表达显着增加了成脂诱导在ADSC中的C /EBPα和PPAR-γ表达以及脂质形成。与对照组相比，miR-150的过表达显着降低了ADSC中Notch3的表达。抑制Notch3促进了ADSC的成脂分化。miR-150可能通过下调Notch3调控ADSC的成脂分化。miR-150还抑制了ADSC的增殖潜能和Nanog的表达。此外,miR-150 下调 Notch3 的表达对 hADSCs 成骨分化具有抑制作用,其机制可能经由Notch3作用FAK/ERK1/2 信号，进而抑制成骨分化。.通过一系列机械、化学和酶学方法对健康人和猪的脂肪组织进行脱脂，保留胶原蛋白和其他成分在脱细胞脂肪组织（hDAT）中。将提取的hDAT制成可注射的水凝胶，在一定条件下其可自组装形成凝胶。hDAT水凝胶对ADSCs无毒，并可在体外自发诱导成脂分化。其具有高度的生物相容性，植入小鼠皮下后不引起炎症和排斥反应。此hDAT水凝胶在采集和植入过程具有无创性。.通过本课题研究，揭示miR-150在ADSCs分化的机制，也为利用ADSC结合脂肪脱细胞外基质修复软组织缺损提供新途径。