lin28/let-7靶向调控Notch信号通路对脂肪干细胞向施万细胞分化作用的研究

Stem cell is known as a ground breaking therapeutic solution for central nervous system disease. We have firstly reported that Schwann cells differentiated from adipose derived stem cells （ADSC–SC）could be a novel and important seed cell source. But in study, we found that the traditional inducing method was low efficiency and instability, poor differentiatied cell maturity. It is important and imperative to clarify the molecular mechanism of the differentiation and then to explore an effective method into induce adipose derived stem cells into Schwann cells in vitro. Studies have shown that miRNAs play an important roles in regulating stem cell differentiation.As so far , there are still no reported about the relationship between miRNAs and differentiation of adipose derived stem cells into Schwann-like cells. In this study, we isolated adipose derived stem cells and induced them differentiatied into Schwann cells. We performed a global miRNA analysis with the application of microaaray assay to reveal and verfied the specific miRNAs expression in differentiation. The result showed let-7 family expression was unregulated obviously. So, we will selecte let-7 as a target and observe the influence of let-7 in differentiation. It will be plan to explore the function of let-7 in the progress of differentiation by gain of function and loss of function experiment which will regulate the expression level of let-7. And we found that Notch signal path was predicted to be the specifc target gene of let-7 by bioinformatics and lin 28 acted as negative regulatory of let-7 biological reccurrence. in order to further investigate the mechanism of let-7 regulation in the differentiation, we will anaylaze the interreaction between lin28/let-7 and Notch signal path. Furthermore, we will compare the two inducing method , tradional method and let-7 lentiviral vector inducing method, by the differentiation efficiency , function of ADSC–SC, and the effect on rat following brain coutusion after tansplanting ADSC–SC. We will discuss the possible mechanism mediated by miRNA in regulating in regulating adipose derived stem cells differentiatied into Schwann-like cells for the first time. It will be help to clarify the mechanism of differentiation of adipose derived stem cells , providing a highly promising therapeutic option for patients with various brain diseases.

干细胞移植为中枢神经系统疾病提供了新的治疗方法。我们曾报道脂肪干细胞来源施万细胞（ADSC-SC）可成为修复脑损伤新的种子细胞。但现有诱导方案面临分化率低且不稳定、分化细胞成熟度差等难题。因此阐明分化的分子机制，寻找更有效促进分化的方法是非常重要的。干细胞分化受遗传严格调控，miRNAs在其中起关键作用；目前尚无研究报道miRNAs与ADSC-SC分化的关系。我们前期利用基因芯片检测大鼠ADSC向SC分化中差异表达的miRNAs，筛选出显著表达上调的let-7。继而拟采用功能获得和缺失实验证实let-7调控分化的作用。再从调控子lin28和靶通道Notch通路两方面深入研究其调控分化的具体机制。并利用let-7诱导分化SC，从细胞功能和移植治疗运动皮层损伤大鼠偏瘫模型两个角度探讨其与传统诱导方式的差异。这为阐明ADSC-SC分化的分子机制提供了重要的依据，对促进其临床应用具有重要的意义。

现有脂肪干细胞向施万细胞分化的诱导方案面临分化率低且不稳定、分化细胞成熟度差等难题。因此我们成功分离、培养脂肪干细胞向施万细胞分化，基因芯片检测大鼠脂肪干细胞向施万细胞分化中差异表达的microRNAs，筛选出表达显著上调Let-7，而同时通过靶基因预测发现Let-7、mir30/34/12等均靶向Notch。因此下一步利用RNA-sqe检测大鼠脂肪干细胞向施万细胞分化过程中差异性基因， RNA-sqe分析发现Notch信号通路中的众多关键基因如Notch3-4, Jag1-2,Hes1都存在差异性表达，KEGG分析提示差异性基因富集在Notch通路。然后通过qRT-PCR和WB验证分化后Notch 1、Jag1和hes1表达显著下调，采用Tangeretin （Notch抑制剂）处理后，促进脂肪干细胞向施万细胞分化，ADSCs的干性基因C-myc和OCT4表达上调，Sox2和KLF4表达下调，这些结果显示Notch通路是脂肪干细胞向施万细胞分化的重要负调控信号通路。同时发现稳定过表达let-7a-5p的ADSCs细胞在同等诱导条件下，更易于分化成施万样细胞，表明过表达let-7a-5p可促进分化；相反地，稳定过表达let-7a-5p-sgRNA的ADSCs细胞在同等诱导条件下，其分化潜能受到了明显抑制。而过表达let-7a-5p可明显抑制RBPJ基因和蛋白的表达，相反地，敲降let-7a-5p则明显促进Rbpj基因和蛋白的表达，提示Rbpj是let-7a-5p的下游靶标之一，let-7促脂肪干细胞向施万细胞分化的作用可能是通过抑制Notch通路实现的。这为阐明脂肪干细胞向施万细胞分化的分子机制提供了重要的依据，对促进其临床应用具有重要的意义。