新基因mgt-16对间充质干细胞增殖、迁移的影响及机制研究

Mesenchymal stem cells（MSCs）represent the most promising candicate cell source for cell replacement therapy, which is one of the feasible strategies in the management of wound healing. However, the molecular mechanisms need to be elucidated. This study is based on a novel gene, mgt-16, cloned and characterizated in murine mesenchymal stem cell line 10T1/2 by gene trap screening, which has been submitted to GenBank (GenBank accession no. GU266552).Our primary results showed its cytoplasmic distribution, its roles in smooth muscle cell differentition, and it may be involed in the process of cell proliferation and migration. In this study, we will construct mgt-16 over-expressed and low-expressed 10T1/2 cell models. The influence of mgt-16 on the cell proliferation will be determined using WST-8 dye (Cell Counting Kit-8), cell migration by scratch wound on monolayer cells, Transwell assay, and skin excision wound model in athymic mouse in vitro and in vivo. The influence of mgt-16 on the cell cycle and apoptosis will be observed by Tunel labeling assay and flow cytometry, and cell proliferation or migration related genes expression will be detected by RT-PCR, immunocytochemistry, and western blot in mgt-16 over-expressed or low-expressed 10T1/2 cells. Finally, the molecular mechenisms of how mgt-16 regulats mesenchymal stem cells proliferation and migration will be elucidated by application of various cell signaling pathways inhibitor or agonist tests. These results would provide theoretical support and therapy strategies in order to more effectively improve wound healing by mesenchymal stem cells.

间充质干细胞（Mesenchymal stem cells，MSCs）是创伤修复中细胞治疗的最佳候选种子细胞，但其增殖、迁移分子机制有待阐明。课题组前期利用基因捕获从MSCs细胞株10T1/2发现一个新基因mgt-16（已提交GenBank，ID号：GU266552)，已研究其细胞分布、与平滑肌分化的关系，并提示可能与细胞增殖、迁移相关。本课题拟建立过表达或沉默mgt-16的10T1/2细胞模型，用CCK-8、细胞划痕、Transwell、裸鼠皮肤切割伤模型等体内外实验进一步确定mgt-16对细胞增殖、迁移的影响；Tunel及流式检测mgt-16对细胞周期、凋亡的影响；RT-PCR、免疫组化和/或Western blot检测增殖及迁移相关基因的表达，并用信号通路阻滞剂/激动剂等手段，揭示mgt-16参与调控MSCs增殖、迁移的分子机制，为MSCs应用于创伤修复提供理论基础和可能的治疗策略。

间充质干细胞（Mesenchymal stem cells，MSCs）是目前组织工程和再生医学研究的热点，对MSCs的增殖、分化、迁移的精确调控是其用于临床治疗前必须解决的问题。我们通过鉴定以C3H/10T1/2细胞(简称10T1/2细胞)作为 MSCs 模型，并建立以LacZ为报告基因的基因捕获阳性克隆库进行研究，获得了一系列结果：证实TGF-β1可诱导10T1/2细胞成平滑肌分化，分离获得4差异表达基因（2个新基因：mgt-6和mgt-16、2个已知基因：Mrps6和Caspase 8)；发现新基因mgt-16为细胞胞浆表达（在核周分布较高）的MSCs增殖和迁移正相关基因；mgt-16低表达可抑制MSCs成脂，mgt-16与MSCs平滑肌分化负相关，同时，可能防止平滑肌过度增殖，在肺癌细胞中高表达；提出了TGF-β1通过激活P38信号通路下调mgt-16基因而调控MSCs平滑肌分化的分子机制；发现Mrps6在MSCs平滑肌分化后表达降低，但在MSCs内皮分化后表达增加；利用microRNA芯片筛选获得了TGF-β1诱导10T1/2细胞平滑肌分化重要的差异microRNA，为研究microRNA调控MSCs平滑肌分化分子机制提供了参考；创新性地提出了SDF-1可通过自诱导、旁分泌及抑制MSCs凋亡等多种机制促进VEGF诱导MSCs内皮分化，为研究MSCs内皮分化提供了新策略。