NeuroD2调控人恶性神经胶质瘤细胞向神经元分化的作用研究

Gliomas are tumors that arise from neuroepithelium, malignant histological types consist 77.5% of all the gliomas. Malignant gliomas are the most common solid tumors in the central nervous system, are essentially incurable due to their rapid growth and very invasive nature. Despite therapeutic improvements through combined neurosurgery, chemotherapy and radiotherapy, current treatment modalities are still unable to significantly prolong patients’ survival. One potential approach is to induce them to become terminal differentiated cells, exciting cell cycle and staying the non-proliferating state. The applicant first reported that the depletion of p53 upregulates neurogenic transcription factors, which in turn boosts fibroblast-neuron conversion. p53 binds the promoter of the neurogenic transcription factor Neurod2 and regulates its expression during fibroblastneuron conversion. In this project we are going to investigate whether NeuroD2 could induce human malignant glioma cells into terminal neurons and whether knockout of p53 could boost this process, whether this conversion is direct or via a “stem/progenitor cell like” stage and what are the telomere length and telomerase change during this process, and what are the gene expression differences among hESC, fibroblast and glioma conversions, so that we can analysis the characterization of glimoas’ conversion and try to find some key genes to improve the induction method.

神经胶质瘤（Glioma）是一种来源于神经上皮的肿瘤，恶性的占所有胶质瘤的77.5%。恶性神经胶质瘤的致命性表现在它的指数增长和侵润性上。如果能使肿瘤细胞分化成终末的神经元，维持在非增殖（或低增殖）状态，降低侵润性，这对于恶性神经胶质瘤的治疗也许是一种可行的辅助策略。申请人在前期的研究中首次发现敲除/敲减p53能促进NeuroD2的表达，使人成纤维细胞向功能性神经元转分化。本课题拟过表达NeuroD2来诱导人恶性神经胶质瘤细胞向终末神经元分化，并观察敲除p53是否能促进这个分化过程；对分化过程是否经历“干/祖细胞”阶段以及肿瘤细胞的自我更新能力和基因组稳定性的改变等进行分析；比较人胚胎干细胞，人成纤维细胞和恶性胶质瘤细胞向神经元分化中的基因表达差异，探索恶性胶质瘤分化的特点，期望寻找到几个关键的差异表达基因，从而进一步改善人恶性胶质瘤细胞向终末神经细胞分化的方法。

恶性神经胶质瘤（Glioblastoma，GBM）的致命性表现在它的指数增长和侵润性上。我们在前期的研究中首次发现敲除/敲减p53能促进NeuroD2的表达，使人成纤维细胞向功能性神经元转分化。本课题建立了敲除p53的U87和U251单克隆细胞系，随机选取其中U87-B7和U251-D3经行诱导分化和其他生物学实验。结果发现U251-D3能向MAP2阳性神经元诱导分化，而U87向MAP2阳性的神经元诱导存在一定阻碍。尤其是过表达NeuroD2能提高U251-D3的诱导分化效率，而U87-B7却基本死亡，提示两者在向神经元分化的能力上存在差异。分化过程可能经历“干/祖细胞”阶段，U251-D3的神经前体标记Pax6、sox2和Vimentin上调。其次，我们比较了U87和U251在敲除前后细胞增殖、侵润、迁移、凋亡、周期等生物学特性。结果显示敲除p53的U87-B7和U251-D3增殖减慢，侵润降低，迁移减少，凋亡增加，周期分布发生改变。将U87-B7和U251-D3移植入裸鼠脑内，未发现肿瘤形成，而移植U87和U251细胞的对照裸鼠肿瘤明显，提前死亡。体内体外实验结果均提示敲除p53可能可以降低GBM的指数生长特性和侵润性。最后课题通过基因表达谱分析，发现Notch3，TGFα和HGF为敲除p53后表达明显下调的基因，其中HGF和Notch可能由p53直接调控。本研究对p53在神经发育过程中的机制研究做了新的探索，可能在神经胶质瘤基因治疗领域有一定应用前景。