水牛骨髓间充质干细胞体外培养发生自我转变的机制及低氧对维持其间质细胞特性的调控作用

Bone marrow mesenchymal stem cells (BMSCs) are a kind of adult stem cells with the characteristic of maintaining self-renewal, proliferation and multiplex differentiation potential, thus BMSCs have become a hot spot of stem cell research in recent years. In our preliminary research work, the primary buffalo BMSCs have been preliminary separated. However, buffalo BMSCs lost the characteristics of mesenchymal cells during in vitro culturing, establishing a stable buffalo BMSCs cell line is still a problem. It has been reported that hypoxic environment is beneficial to maintain the self-renewal properties of stem cells. Therefore, in this study, firstly, the changes of buffalo BMSCs mesenchymal properties during in vitro culture under normoxia environment will be investigated, moreover, the expression level of mesenchymal markers and their transcription factor twist, HIF in the buffalo BMSCs treated with siRNA and overexpression of HIF will also be analyzed to reveal the molecular mechanism of MET process. Meanwhile, the regulation function of hypoxic environment on the maintenance of mesenchymal properties in buffalo BMSCs will also be investigated to reveal the molecular mechanism of MET converting into EMT during in vitro culture of buffalo BMSCs under hypoxic environment. These works will be benefit for enriching the regulatory network research of self-renewal in buffalo BMSCs, so that to optimize the culture system of buffalo BMSCs, and successfully establish a stable culture system for buffalo BMSCs.

骨髓间充质干细胞（BMSCs）是一类具有维持自我更新、增殖和多向分化潜能特性的成体干细胞，成为近年来干细胞研究的热点。本课题组前期工作已初步分离出原代水牛BMSCs，但经体外培养会随着代数的增加而渐渐分化，很难建立稳定的水牛BMSCs细胞系。研究表明低氧利于干细胞多能性的维持。为此，本项目将在现有工作基础上，通过研究水牛BMSCs在常氧环境下传代培养过程间质细胞特性的变化，并通过构建siRNA干涉和过表达HIF的BMSCs细胞，分析BMSCs 间质标记基因及其转录因子的表达变化，探讨BMSCs培养过程中发生间质上皮转变（MET）的分子机制；同时，系统研究低氧环境对水牛BMSCs间质细胞特性维持的调控作用，进一步揭示低氧环境触发BMSCs从MET到EMT（上皮间质转变）逆转作用的机理。这将有助于丰富和完善水牛BMSCs细胞自我更新的调控网络，对建立稳定的水牛BMSCs培养体系具有重要意义。

本项目探讨了水牛胎儿间充质干细胞（BMSCs）体外分离培养的相关技术环节对其分离效果及间质细胞特性的影响；同时研究了水牛BMSCs在常氧环境下传代培养过程间质细胞特性发生转变的情况以及低氧环境对BMSCs细胞间质特性维持的调控作用。首先比较了不同的胎龄和不同血清浓度等因素对水牛胎儿BMSCs分离培养效果及其间质细胞特性的影响，成功分离得到的水牛胎儿BMSCs具有间充质干细胞的特性，建立了稳定的水牛胎儿BMSCs分离培养方法。其次，通过研究水牛BMSCs在体外常氧环境下传代培养过程间质细胞特性的变化情况，明确了BMSCs在体外传代培养过程中细胞会随着培养代数的增加细胞发生MET从而失去间质细胞的特性；同时，探讨了低氧环境（5%O2）对水牛BMSCs细胞间质特性维持的调控作用，发现低氧培养可以促进BMSCs细胞发生上皮间质转变（EMT），从而有助于维持BMSCs间质干细胞的特性。进而，探索了低氧培养条件下lncRNA H19对水牛BMSCs细胞间质特征的影响，初步阐明了低氧培养条件可促进水牛胎儿BMSCs lncRNA H19的表达，而且lncRNA H19是通过PI3K/AKT通路来调控水牛BMSCs 发生EMT，从而对维持BMSCs的间质特征起着重要的促进作用。另外，还分析了不同性别水牛胎儿BMSCs细胞中印记基因Xist甲基化状态，揭示了水牛BMSCs印记基因Xist甲基化状态与性别有关。这些结果为获得稳定的水牛骨髓间充质干细胞系及其后续的应用提供了有力的理论支撑。