双酚A暴露对神经嵴干细胞良恶性分化潜能的影响

Neuroblastoma(NB) is one of the most common solid malignant tumors in children.NB derives from neural crest cells (NC) which can not be differentiated normally and spin off cancer stem cells during the embryo development. However, little is known about the risk factors and their mechanisms. Environmental endocrine disruptors (EED) are recently identified risk factors associated with various human diseases. Our previous studies have implicated the role of EED including bisphenol A (BPA) in neuroblastoma cell proliferation, multidrug resistance, migration and invasion. The estrogen receptorβ（ERβ）-dependent pathway may contribute to the underlying mechanisms. Here, we further investigated the effects of BPA exposure on both benign and malignant differentiative potential of neural crest stem cells. Normal human induced pluripotent stem cells (hIPS) were differentiated into NC in vitro. Dose-time effects of BPA on the differentiation of NC into sympathetic neurons were detected by the confocal microscopy. BPA-induced malignant differentiative potential of NC into neuroblastoma stem cells was evaluated by flow cytometry, soft agar clonogenic and tumorigenesis assays. ERβ mRNA and protein expressions were assessed by real-time PCR and western blot analysis. The association between toxic effects of BPA and ERβ expression levels was also investigated.This study will explore the role of BPA in NB oncogenesis and elucidate the pathogenesis of NB with the goal of creating improved precautions for this type of tumor.

神经母细胞瘤(NB)为婴幼儿最常见恶性实体肿瘤之一。胚胎期神经嵴干细胞（NC）向外周交感神经元的良性分化受阻及向肿瘤干细胞恶性转化是NB发生的关键过程，诱因及机制尚不明确。环境内分泌干扰物（EED）是新近发现威胁人类健康的致病因素。申请者前期研究发现双酚A（BPA）等EED通过β-雌激素受体（ER）介导可促进NB进展恶化。本课题拟体外诱导人多能性干细胞分化为NC，采用激光共聚焦显微技术观察不同浓度BPA对NC向交感神经元体外定向分化的影响；应用流式细胞分选技术、体外细胞克隆形成实验及裸鼠移植瘤模型观察BPA在NC向NB肿瘤干细胞恶性转化中的作用；应用荧光定量PCR及免疫印迹分析BPA对ERβ表达的影响，并运用RNAi等技术分析ERβ不同表达水平对BPA作用的影响。揭示BPA在NB发生中的作用及机制，阐明EED与NB发生的相关性，为NB的早期预防及相关公共卫生政策制定提供依据。

神经母细胞瘤(NB)为婴幼儿最常见恶性实体肿瘤之一。胚胎期神经嵴干细胞（NC）向外周交感神经元的良性分化受阻及向肿瘤干细胞恶性转化是NB 发生的关键过程，诱因及机制尚不明确。环境内分泌干扰物（EED）是新近发现威胁人类健康的致病因素。申请者前期研究发现双酚A（BPA）等EED 通过β-雌激素受体（ER）介导可促进NB 进展恶化。本课题拟进一步探讨BPA对神经嵴细胞良恶性分化的影响及作用机制。应用体外细胞试验诱导人多能分化干细胞（IPS）分化形成神经嵴细胞及外周交感神经元，建立干细胞向神经嵴细胞及外周交感神经元分化的体外细胞实验模型。运用BPA体外干预实验观察BPA对神经嵴细胞向外周交感神经元定向分化的影响，发现对照组与不同浓度BPA 组之间分化诱导后细胞形态学上无明显差异，检测各组诱导分化后的细胞中p75神经嵴细胞标记物及β-tubulin外周交感神经元标记物表达无明显差异（P > 0.05）。进一步观察BPA与雌二醇（E2）共同作用对神经嵴细胞向外周交感神经元定向分化的影响，发现对照组与E2及BPA干预组之间分化诱导后细胞在形态学上无明显差异。检测发现神经嵴细胞向外周交感神经元定向分化过程中，细胞分化调控基因TGFA/TGFα表达量明显增高（P<0.05）。BPA与E2共同作用下可明显抑制TGFA/TGFαmRNA及蛋白水平的表达（P<0.05）。同时观察BPA在人神经嵴细胞向神经母细胞瘤干细胞恶性转化中的作用，检测发现BPA干预组分化获得细胞中c-kit与CD113等神经母细胞瘤干细胞分子标记物表达水平较对照组明显升高（P>0.05）。体外细胞克隆实验发现BPA干预组原代及二代细胞形成克隆能力较对照组明显增强。但体内细胞成瘤实验未见BPA干预后分化形成的外周交感神经元细胞具有成瘤能力。通过病毒转染建立β-ER抑制表达的神经嵴细胞，发现β-雌激素受体表达下调＋BPA干预组细胞中c-kit与CD113等神经母细胞瘤干细胞分子标记物表达水平较对照组无明显升高（P>0.05）。本项研究初步提示BPA具有一定的干扰神经嵴细胞向外周交感神经元细胞正常分化及促进其向神经母细胞瘤干细胞异常恶性分化的作用，而这一作用主要是通过β-ER途径实现的。初步揭示环境内分泌干扰物在NB发生中扮演了重要角色。