研究显示肝癌CD133阳性细胞具备肿瘤干细胞特征，是临床治疗的靶标。我们前期发现高尔基体蛋白SCYL1BP1 在肝癌中低表达且与肿瘤分化程度正相关；该基因在肝癌CD133阳性细胞中表达明显低于CD133阴性细胞；敲减CD133阴性细胞SCYL1BP1表达可致OCT4表达上调、细胞增殖能力增强。提示该基因可能负调控肝癌CD133阳性细胞的生物学特性，但其机制不明。本项目拟首先应用基因导入或敲减技术，分别观察SCYL1BP1导入或沉默后对肝癌CD133阳性或阴性细胞自我更新、分化、增殖和体内成瘤等肿瘤干细胞特征的影响；进一步通过RNA-seq和免疫共沉淀等蛋白质组学技术，探讨筛选该基因导入或沉默后涉及的特异性信号分子及其通路，并在大样本肝癌组织芯片中验证；最后在肝脏条件性SCYL1BP1基因敲除小鼠模型中，确证该基因在肝癌发生发展中的重要作用。项目的实施有望为肝癌的肿瘤干细胞治疗提供新思路。

It was reported that CD133 positive hepatocellular carcinoma (HCC) cells had the characteristics of cancer stem cells (CSCs) and may be the target of future tumor therapy. Our previous study showed that SCYL1BP1, a golgin, was down-regulated in HCC samples. Expression of SCYL1BP1 was positively associated with differentiation grade of HCC. Futhermore, the expression level of SCYL1BP1 was much lower in CD133+ HCC cells than that in CD133- HCC cells. Knocking down SCYL1BP1 in CD133- HCC cells upregulated OCT4, a critical gene in stem cell self-renewal; and promoted tumor cell proliferation. These data suggested that SCYL1BP1 negatively regulated biological features of CD133+ cells, while the detailed molecular mechanism remains unclear. In this study, we will try to conditionally overexpress/knockdown SCYL1BP1 in CD133+/- HCC cells, and investigate the effect on characteristics of CSCs both in vitro and in vivo, such as self-renewal, differentiation, and proliferation. Furthermore, by using RNA-seq and co-immunoprecipitation based-proteomics, we will screen the potential down-stream signaling pathways which are altered both in overexpressing and in knocking-down experiments and verified by immunohistochemistry in HCC tissue microarray. Finally, we will investigate the role of SCYL1BP1 in HCC carcinogenesis by establishing and using liver conditional SCYL1BP1 knockout mice model. Hopefully our research will offer a new way for future HCC CSC therapy.

项目组对SCYL1BP1在干细胞的调控研究中发现，其上游基因CHD1L可能与干细胞的干性维持和多能性有一定关系。我们在人胚胎干细胞（hESC）中过表达CHD1L发现其外胚层尤其是PAX6表达上调，进一步培养发现其向神经外胚层分化；相反，敲减hESC中的CHD1L会损害其向神经外胚层的分化。动物实验中发现高表达PAX6的胎鼠脑室区域同样表达CHD1L。在hESC向造血/内皮诱导过程中我们有以下发现：过表达c-MYC的hESC在半固体培养基中多能性基因表达下调，造血相关基因表达升高，红系标志（CD71和CD235a）表达增加。而在红系定向诱导培养基中红细胞的标志基因变化却不明显，以上数据显示c-MYC在hESC向造血细胞分化尤其是红细胞分化起到一定的作用；过表达FLI1基因可以将约60%的细胞定向分化为CD34+/CD38-造血相关细胞。我们在过表达FLI1基因的同时激活PKC通路，可以在3天内得到90%以上的细胞表达CD31+/144+（内皮细胞标记）。检测发现这些细胞具有内皮的特征并表达内皮功能。我们在这一内皮诱导过程中运用siRNA分别沉默PKC的9个亚基，发现PKCε和PKCη的沉默显著降低了内皮的生成，前期测序的结果也支持这一现象。通过运用PKC抑制剂和VEGF的siRNA，我们发现在内皮生成的过程中VEGF发挥一定作用，而且是通过PKC通路来实现的。血小板无力症为血细胞对多种生理诱聚剂反应低下或缺如。我们利用患儿的皮肤成纤维细胞构建了iPSC细胞系（GT-iPSC）并将之成功诱导为血小板，检测发现血小板激活标记（PAC）阴性，也不能够聚集，血小板功能缺失，说明我们建模成功。在此细胞系中过表达ITGA2B基因可以纠正缺陷血小板的表型，表明血小板无力症的基因治疗可能是有效的，为这一疾病的治疗带来了证据和希望。现有FISH-PGD或者芯片/测序进行PGS后仅能区分非平衡胚胎，而无法对携带结构异常染色体的平衡胚胎和正常胚胎进行植入前鉴别。我们运用MicroSeq技术（染色体显微切割技术与新一代测序技术相结合）成功解决了此难题，降低后代遗传病风险，使得这部分患者的后代可以摆脱反复流产或者不孕不育的风险。