层状双氢氧化物对小鼠胚胎干细胞自我更新与分化的影响

Embryonic stem cells(ESCs), which have two unique characteristics: self-renewal and pluripotency, are thought to hold great promise for regenerative medicine. However,the tedious process，the presence of animal components and higher costs of embryonic stem cell culture has been a major challenge today. Recently, there is accumulating evidence that nanotechnology has the potential to solve this problem because of the effects of their structure and properties on the self-renewal, proliferation and differentiation of stem cells. Our previous studies have demonstrated that Layered double hydroxide (LDH) nanoparticles can promote self-renewal and inhibit differentiation of mouse embryonic stem cells(mESCs), which deserves further research. In this study, LDH with different particle size will be synthesed. The cellular effects of LDH on the self-renewal and diffenrentiation of mESC will be evaluated by the comparison of morphology change, alkaline phosphatase activity, and measure the expression of molecules involved in the self-renewal and differentiation of mES cells. In addition, the pluripotency of LDH-treated mESCs will be also assessed by formation of embryoid bodies,teratomas and chimeric Animals. From the results, the most effective LDH nanomaterial will be selected. What's more, the expression change of receptor in mechanotranduction will be evaluated to verify the involvement of mechanical stimuli. Meanwhile, the expression of genes and proteins related to pathways stimulated by mechanical stress will be also measured to identify the molecular mechanism. Our research will reveal how nanoparticles impact the fate of stem cells, which will be important for the rational design of new nanomaterials for tissue engineering and other biomedical applications.

胚胎干细胞具有自我更新和多能性的特征，为再生医学领域带来了广阔的应用前景，但胚胎干细胞培养过程工序繁琐，存在动物组分且成本较高等，是目前所要解决的重大问题。研究表明纳米材料因其独特的结构和功能对干细胞的自我更新、增殖和分化具有较大的影响，是解决上述问题的一条有效途径。本课题组初步研究发现层状双氢氧化物（LDH）能够促进小鼠胚胎干细胞（mESC）的自我更新，值得进一步研究。本项目拟合成不同粒径的LDH纳米材料，通过比较mESC表面形态，碱性磷酸酶活性，自我更新和分化相关基因的表达变化，筛选出最佳促进mESC自我更新的LDH纳米材料，并通过拟胚体、畸胎瘤和嵌合体形成实验评价细胞多能性。同时，通过检测机械传导受体及其介导的相关机械传导通路上基因的表达，明确LDH是否是通过机械刺激对mESC产生作用及其分子机制。为揭示纳米材料如何调控干细胞的命运，开发应用于组织工程学和生物医学的新型材料奠定了基础。

层状双氢氧化物是一种阴离子水滑石样化合物，生物相容性好，低细胞毒性，已广泛用于生物医学领域。本课题研究了LDH 纳米颗粒对小鼠ESCs的影响，揭示了LDHs调控干细胞的作用以及在干细胞研究和干细胞应用中的潜能。.主要研究内容和结论如下：.（1）运用水热法合成了粒径大小约为100 nm的LDH NPs。对mESCs生存率、乳酸脱氢酶释放、活性氧的产生以及细胞凋亡进行检测，评价了LDH NPs与mESCs的生物相容性。结果显示，40 μg/mL以内的LDH NPs对mESCs不产生明显的毒性作用，具有良好的生物相容性。.（2）检测LDH NPs对mESCs自我更新能力的影响。LDH NPs能够促进无饲养层培养条件下mESCs碱性磷酸酶含量，上调多能基因Nanog、Oct4、Sox2基因的表达，并在长期培养过程中依然能够维持细胞的自我更新能力。在进一步去除白血病抑制因子的情况下，LDH NPs能够抑制mESCs的自发分化，下调胚层相关基因的表达，同时维持多能基因表达水平，说明LDH NPs对ESCs自我更新能力的促进作用是不依赖于LIF存在的。.（3）检测LDH NPs处理的mESCs在体内和体外的分化能力。LDH NPs处理的mESCs在体外能够形成拟胚体，继续分化后表达三个胚层特异性基因；细胞皮下注射到免疫缺陷小鼠，能够形成由三个胚层组织构成的畸胎瘤。.（4）研究LDH NPs促进mESCs自我更新的分子机制。通过对自我更新相关通路的检测，发现LDH NPs能够激活PI3K/Akt信号通路。运用PI3K抑制剂能够阻止LDH NPs对Oct4基因的上调，证明了LDH NPs是通过PI3K/Akt通路对多能基因进行调控。.（5）分析了LDH NPs粒径大小对mESCs生物学功能的影响。利用水热法和共沉淀法制备了粒径大约为100 nm、50 nm和30 nm的 LDH NPs，比较了它们对mESCs的毒性作用、细胞内吞和自发分化过程的影响。.（6）检测LDH NPs对mESCs上皮-间质转化的影响。LDH NPs能够调控mESCs自发分化过程中EMT相关基因的表达，维持E-cadherin的表达，抑制N-cadherin、Snail和Slug的升高，并阻止ESCs自发分化引起的细胞骨架的重塑，从而抑制EMT过程。