分区式人工脊髓导管体内定位诱导反应性星形胶质细胞直接重编程神经元修复脊髓损伤

It is well recognized that two major pathological changes disable neuroregeneration after spinal cord injury (SCI), i.e. neuronal loss due to cell death including apoptosis, and an unfavorable regeneration microenvironment such as glial scar resulted from activation and proliferation of astrocytes. Trying to solve these two challenging tasks so as to regenerate the injured spinal cord, the majority of experimental studies have been focused on the two aspects separately; either supplementing neurons via cell transplantation or improving regeneration milieu by inhibition of astrocytic proliferation or by degradation of glial scar formed, and exhibited unfavorable repair outcomes. However, no reported therapeutic studies have been tried so far to target neuronal loss and astrogliosis simultaneously so that one can take advantage of one part to counter the other. In light of this interesting yet unaddressed issue, we hereby propose to apply a dual-aimed repair strategy for SCI. Our hypothesis is that by using the direct reprogramming technology which we established previously and a refined recipe of cocktail transduction factors, reactive astrocytes can be induced directly in vivo into neurons, which will not only constitute a neuroregeneration basis by compensating neuronal loss but also improve regeneration microenvironment by reducing the number of reactive astrocytes and thus the amount of glial scar formation. We will deliver retroviruses carrying transduction factors-encoding sequences to reactive astrocytes in the gray matter area by incorporating the viruses in corresponding compartment of the partition-mimicking multichannel tubular scaffold, which we developed in the previous project and will be implanted into severed spinal cord. We will then apply methods of molecular and cellular biology and neurobiology to assess the efficacy of direct neuronal induction, and to evaluate the repair outcomes of spinal cord injury. The implementation of this project is anticipated to help establish a new approach for SCI therapeutics.

脊髓损伤后，神经元会出现死亡或凋亡，使数量减少；同时胶质细胞的活化及增生，又形成胶质疤痕等不利轴突再生的微环境；这些均是共认的阻碍损伤脊髓顺利修复的不利因素。针对这两个棘手难题，多数研究仅是孤立地通过移植细胞补充神经元数量，或通过抑制胶质细胞增生、分解胶质疤痕，改善修复微环境。但能否有一种同时兼顾这两个方面，并以夷制夷的一举两得手段？值得人们探究。本课题设想：通过重编程技术，选择最佳转录因子组合，诱导反应性星形胶质细胞在体内直接转化为神经元，这样既可以补充丢失的神经元，提供神经再生的物质基础；又能降低胶质细胞数量，避免胶质疤痕过度增生，改善神经再生的微环境。并借用“分区式人工脊髓导管”的灰质区域释放逆转录病毒定位感染反应性星形胶质细胞，转录因子组合诱导其部分原位直接重编程神经元，并以相应的分子、细胞和神经生物学等技术体内验证修复效果。此研究成功将可能为损伤脊髓治疗开辟一个新思路和新方法。

能否寻找到脊髓损伤后，既能兼顾抑制星形胶质细胞的活化、增生，并形成胶质疤痕的不利微环境，又能补充因死亡或凋亡引起数量减少的神经元，产生以夷制夷、一举两得的效果？本课题就此设法通过重编程技术，选择转录因子组合（AscL1、Myt1L、POU3F2、ISL1），或小分子组合（SB431542、LDN-193189、RA、bFGF、Purmorphamine、Forskolin、VPA），诱导反应性星形胶质细胞直接重编程转化为神经元，这样既可以降低胶质细胞数量，避免胶质疤痕过度增生，改善神经再生的微环境；又能以星形胶质细胞转化来补充丢失的神经元，提供神经再生的物质基础。在体内的实验中，有部分星形胶质细胞在转录因子的组合作用下转化成神经元，当然由于转变的数量还不高，故脊髓损伤后的功能恢复并不明显。与体外的转录因子组合诱导直接重编程相比，小分子药物组合诱导神经元表达Tuj1、MAP2和NeuN阳性，其中MAP2阳性率能超过80%，且与MAP2共标的细胞中超过90%能同时表达HB9和CHAT。另外，也尝试了大鼠源转录因子针对大鼠源星形胶质细胞、人源转录因子组合对人源星形胶质细胞进行了同源诱导的初步实验，观察同源和异源的诱导效果差别，结果表明差异并不明显。同时，也进行了人骨髓源的多系分化持续应激细胞在组织工程学的种子细胞治疗神经系统损伤的可能性探索。结果表明诱导的多系分化持续应激细胞源的NPCs具有神经干细胞的特性，能向三种神经组织细胞分化，也具有一定的生物学功能。将此来源的NPCs植入到损伤脊髓节段，同样能够分化成神经元、少突胶质细胞，可以调节局部微环境，降低反应性星形胶质细胞的活性，有利于神经纤维向损伤区域延伸。另外，在已制备的分区式人工脊髓导管工艺上又进行了优化，使尺寸更加符合180-200克的大鼠的脊髓修复使用；优化的导管理化特性、物相容性试验，均表明是良好。还对miR-17在脊髓损伤中的作用机制进行了探索，表明少突胶质细胞分泌的miR-17-5p通过外泌体被星形胶质细胞摄取，再调控IRF1、EGR2、EGLN3和TLR4等靶基因，进而引起靶细胞的活化增殖。这些研究结果将可能为损伤脊髓治疗寻找新思路和方法。