维甲酸联合GSK3和TGFβ受体抑制剂诱导人胚胎干细胞分化为后脑神经干细胞及其脊髓损伤治疗作用研究

Spinal cord injury (SCI) is a serious invasive disease of the central nervous system with high mutilation rate, and caused great distress to people's living and working. The conventional treatment, such as large doses of glucocorticoid hormones, surgical treatment cannot be a fundamental solution to the regeneration of neurons. Transplantation of neural stem cells (NSCs) can effectively improve the recovery of damaged spinal cord, having became the focus in the field of SCI therapy. However, the lack of transplantable NSCs has limited the development of this therapy. The embryonic stem cells (ESCs) can proliferate indefinitely and is expected to provide a valuable cell source of various diseases and injury. In our previous study, for the first time, we found that the joint induction of the inhibitors of GSK3 and TGF beta signaling pathway (CHIR99021, and SB431542) induced ESCs to differentiate into the NSCs with the characteristic midbrain identity. In addition, this NSCs can be amplified in long-term and maintain phenotypic stability in vitro. We assume that on the basis of the original protocol, we could directly obtain the NSCs with the hind brain characteristic by adding the high concentrations of retinoic acid during induction. This proposal intends to apply this protocol to induce ESCs differentiate into the NSCs with the hind brain characteristic, and study their role in the therapy of spinal cord injury in animal models. The study could provide a sufficient source of cells for the stem cell transplantation in SCI.

脊髓损伤(SCI)是一种严重的中枢神经系统创伤性疾病，致残率高，给人们生活和工作带来很大困扰。传统治疗手段如大剂量糖皮质激素、外科手术治疗等不能从根本上解决神经元的再生问题。神经干细胞（NSC）移植可有效改善受损脊髓的恢复，成为SCI治疗研究的焦点，但是NSC来源有限是该疗法发展的瓶颈。胚胎干细胞(ESC)具有多能性，并可无限增殖，体外诱导其向神经分化有望为神经干细胞移植提供稳定的细胞来源。项目组成员的前期研究首次发现联合GSK3和TGF beta信号通路的抑制剂（CHIR99021和SB431542）可将hESC诱导分化为具有中脑特性的NSC，且该NSC可在体外长期扩增并维持表型的稳定。我们假设在原有方案的基础上添加高浓度维甲酸，直接获得具有后脑特性的NSC。本课题拟采用该方案获得后脑特性的NSC，并研究其对脊髓损伤动物模型的治疗作用。该研究的开展将为SCI的神经干细胞治疗提供重要参考。

背景：神经干细胞移植（Nueral Stem Cells, NSCs）可有效促进脊髓损伤神经功能的恢复，来源有限是该疗法发展的瓶颈。胚胎干细胞（Embryonic Stem Cells, ESCs）具有多能性，可体外分化为神经干细胞，为其提供稳定的细胞来源。.方法：1.人胚胎干细胞复苏传代，约30%融合时更换神经诱导培养基（2uM DMH-1, 2uM SB431542和1uM RA），连续培养6天。形态学观察结合RT-PCR及免疫荧光检测神经干细胞和后脑标志。神经干细胞继续分化，检测其分化潜能。2.成年雄性SD大鼠20只，建立全横断脊髓损伤模型，损伤后1d、3d、1W记录BBB评分。一周后取12只大鼠蛛网膜下移植人源性神经干细胞，移植细胞浓度为5*107/ml，体积为10μl。4只注射等量培养基，4只不做处理。移植后1W、2W、3W、4W进行BBB评分。移植4W后处死动物，取出损伤部位含上下各1cm左右长度脊髓，行HE染色。.结果：1. 诱导的细胞神经干细胞标志N-cad、Nestin、Pax6及后脑标志HOXA2、HOXA4、HOXB4均呈阳性，神经干细胞继续分化后可得到不同数量的神经元和胶质细胞。2.模型组大鼠造模后1d、3d、7d BBB评分显著降低，随时间延长BBB评分增长缓慢，表明该模型脊髓的自我修复能力不明显；培养基对照组与模型组相比无显著差异（P>0.05）；干细胞治疗组移植干细胞后1w BBB评分显著增高（P<0.05），但2w、3w、4w时与模型组无显著差异（P>0.05）。HE染色镜下观察可知：各组别组织切片均见脊髓组织白质区神经元变性、坏死，脱髓鞘至空洞形成；灰质区组织大片坏死、崩解，结构消失，并伴随较多巨噬细胞浸润并累积至白质区。其中模型组和培养基对照组组织崩解面积较多，干细胞治疗组组织崩解面积相对较少。.结论：本研究采用的方案可将人胚胎干细胞诱导为后脑表型的神经干细胞，且该细胞对大鼠脊髓损伤早期具有一定的修复作用。这一研究的开展为神经干细胞移植提供了稳定的细胞来源，为脊髓损伤干细胞治疗提供重要参考。