高表达VX15/2503抗体毛囊神经嵴干细胞载体的构建及对神经胶质瘤的作用与机制研究

We found in our previous study that "SEMA4D, an axon guidance molecules and it's receptor Plexin-B1 are overexpressed in glioma, and involved in tumor invasion and angiogenesis of glioma" ,It is known that the monoclonal antibody VX15 / 2503 can block the binding of SEMA4D to its receptor, but how to establish a high expression vector with tumor chemotaxis and its inhibitory effect on glioma has not been reported yet; meanwhile, we found that Hair Follicles Neural Crest Stem Cell (hfNCSC) could be used as a vector of gene therapy ,and exocrinosity of monoclonal antibody VX15/2503 of SEMA4D by transgenic hfNCSC was confirmed after the hfNCSC is transfected by lentivirus. So, we proposed that "high expression of VX15 / 2503 by transgenic hfNCSC blocks the binding of SEMA4D to Plexin-B1, regulates apoptosis, migration and angiogenesis ，then inhibits the proliferation and invasion of glioma. Glioma cell lines U87MG and U251 as well as the nude mice model will be used in our study, VX15/2503 monoclonal antibodies will be expressed by gene editor modified hfNCSC , cell culture and stereotactic injection method, application of precipitation, the fluorescence localization and path analysis technology will be used to explore the expression of transgenic hfNCSC antibody as well as the mechanism of proliferation, invasion, the action of angiogenesis to glioma to provide a new precision therapy of glioma.

课题组前期发现SEMA4D及其受体Plexin-B1在胶质瘤细胞中过表达且参与肿瘤增值及侵袭。已知单克隆抗体VX15 /2503可阻断SEMA4D与其受体结合,但如何建立向肿瘤趋化的高表达载体及对胶质瘤抑制效果未见报道。近期我们发现毛囊神经嵴干细胞（hfNCSC）可作为基因治疗载体;通过慢病毒转染初步证实VX15/2503能被转基因hfNCSC外分泌。据此我们提出假说:转基因hfNCSC高表达VX15/2503有效阻断SEMA4D 与Plexin-B1结合,调节细胞凋亡、迁移及血管生成,抑制胶质瘤增殖和侵袭。课题拟以U87MG、U251胶质瘤细胞株及裸鼠胶质瘤模型为研究对象,利用基因转染修饰hfNCSC表达单克隆抗体,通过细胞共培养及立体定向注射移植到瘤区;应用免疫沉淀、荧光定位及通路分析技术,探讨转基因hfNCSC对胶质瘤细胞增值、侵袭、血管生成的作用及机制，为胶质瘤精准治疗提供新方法。

脑胶质瘤是最常见的原发性颅内恶性肿瘤，具有高致死及易复发性等特点，是当今医学界亟待解决的难题。本项目针对胶质母细胞瘤易复发、预后差的特点以及治疗手段有限的现状，结合课题组前期研究的发现，成功筛选出高表达VX15 /2503 的转染毛囊神经嵴干细胞；确定重组人源化单克隆抗体VX15/2503蛋白序列，完成重组人源化单克隆抗体的制备；摸索出最适毛囊神经嵴干细胞生长的低氧条件，并鉴定出低氧条件下的毛囊神经嵴干细胞特征性基因和蛋白表达改变；探究影响毛囊神经嵴干细胞在不同氧浓度下增殖及分化潜能差异的机制，发现在低氧情况下，Hippo信号通路和Wnt信号通路参与了毛囊神经嵴干细胞的增殖与分化的调节。研究发现毛囊神经嵴干细胞可在胶质瘤周低氧、低pH微环境中适宜生存，其具有趋瘤性富集于胶质瘤旁，探究低氧条件影响毛囊神经嵴干细胞的增殖及分化的具体机制，为脑胶质瘤精准治疗提供了新的思路。依托课题研究，课题组培养硕士研究生2人，博士研究生3人，博士后1人。研究成果发表SCI Q1区论文2篇，中文核心期刊论文1篇。举办和参加国内外成果交流会议13次，和同行进行充分交流，取得良好效果，课题研究成果为脑胶质瘤精准治疗提供新的思路，具有良好的临床转化前景。