Bmal1和Clock对胚胎干细胞致瘤性的调控及其分子机制

Tumorigenicity is one of the biggest hurdle for the clinical application of Embryonic stem cells in regenerative medicine. However, the underlying mechanism is mainly elusive. It is believed that c-MYC, p53 pathway and Wee1 play critical role for the ES related tumorigenicity and differentiation. Of note, all of these factors are regulated by transcription factors Bmal1 and Clock which can form heterodimer to regulate the expression of a large amount of genes in the adult somatic cells and adult stem cells. It indicated that both Bmal1 and Clock are expressed in mouse embryonic stem cell. However, their expression in ES cells are heterogeneous, i.e. some cells express both factors, some cells express one of them, and some of cells are negative for both of them. In addition, transient transfection of Bmal1/Clock results in lower proliferation rate in ES cell. Thus, it is reasonable to speculate that individual cells with Bmal1/Clock heterodimer can regulate the expression of c-MYC, p53/p21 and Wee1, while the cells without Bmal1/Clock can not, which is very likely to result in the difference in forming tumor. In the present study, we are going to establish a method to collect ES cells based on their presence or absence of Bmal1 and Clock. And then we are going to examine the difference in the pluoripotency and tumorigenicity for different cell populations. In addition, expression and function of c-MYC,p53/p21, Wee1 and possible other Bmal1/Clock targets will be examined. This study will provide new insight in the understanding of molecular basis of ES related tumorigenicity and differentiation.

致瘤性是胚胎干细胞（ES）走向临床应用的巨大障碍，但其分子机制仍不清楚。目前认为，c-MYC、p53/p21、Wee1等节点对于ES致瘤性发挥重要调控作用。成体细胞中这些节点均受转录因子Bmal1/Clock调控。我们的研究表明，Bmal1/Clock在ES中表达，且具有异质性。同一ES细胞系中部分细胞同时表达两因子，部分细胞仅表达其一，甚至均不表达。此外，瞬时转染Bmal1/Clock使ES增殖速度显著下降。据此我们推测，Bmal1/Clock表达差异的细胞群中，c-MYC、p53/p21、Wee1的表达也存在差异，由此可能导致不同细胞群间致瘤性的差别。本研究将建立筛选存在Bmal1/Clock表达差异的ES细胞群的方法，分析各细胞群在致瘤性上的差别。以及在ES细胞中Bmal1/Clock对c-MYC、p53/p21、Wee1及其他靶点的调控。本研究有望为理解ES致瘤分化提供新的视角。

我们前期的研究表明Bmal1/Clock 在ES细胞中表达且呈明显异质性。同一细胞株中，部分细胞同时表达两因子，部分细胞仅表达其一，甚至均不表达。据此我们推测，Bmal1/Clock 表达差异的细胞群中，c-MYC，p53/p21,Wee1 的表达也存在差异，由此可能导致不同细胞群间致瘤性上的差别。本研究目标在于筛选存在Bmal1/Clock 表达差异的ES 细胞群的方法，分析各细胞群在致瘤性上的差别。以及在ES 细胞中Bmal1/Clock 对c-MYC,p53/p21，Wee1 及其他靶点的调控。.通过本研究，我们成功建立了筛选存在Bmal1表达差异的ES细胞群的方法，分析了不同细胞在生长、分化、致瘤性上的差异。并进一步分析了下游c-MYC, p53/p21，Wee1的表达情况。从研究意义上看，一方面是建立了根据Bmal1表达与否筛选细胞的技术体系。Bmal1是时钟基因中最为核心的一个，目前已经发现和昼夜节律的调节、细胞增殖分化、氧化应激反应等重要生理过程密切相关。国际上多个实验室已经建立了Bmal1转录、翻译、波动监测的研究体系，但是根据Bmal1表达与否筛选细胞的技术体系尚无报道。本实验室在这一方向填补了空白。另一方面是利用上述研究体系发现Bmal1与p53及其通路上重要节点基因间存在着调控作用，并且这一作用能够影响细胞的增殖和ES细胞的致瘤性。这一结果为认识ES细胞的致瘤作用提供了新的方向。