Tet3质粒诱导HUCMSC分化为神经干细胞复合PLCL/CA-SF三维纳米神经导管支架治疗脊髓损伤的实验研究

Although axon is renewable after spinal cord injury, it couldn’t grow across the scar interface effectively, which becomes a major problem influencing the treatment of spinal cord injury. Our preliminary study has confirmed that neural stem cells could be differentiated from human umbilical cord mesenchymal stem cells (HUCMSCs) induced by small molecular protein, but its efficiency was not high enough, and usually difficult to replicate. Many genes promote the progress of the occurrence, differentiation and maturity of nerve tissue. For instance, overexpression of Tet3 could get mature neurons more effectively. However, for lack of effective support, the curative effect of the repairment of injuried spinal cord is not good enough with the single transplantation of neural stem cells. Therefore, we speculate that if we can gain neural stem cell more effectively and find a kind of satisfactory sustentaculum which can provide a condition for the cells to adhere and proliferate, the injuried spinal cord could be repaired better. In this study, we first induce human umbilical cord mesenchymal stem cells to differentiate into neural stem cells by overexpression of Tet3; Secondly, constructing PLCL/CA-SF three-dimensional multichannel nerve guidance conduit with supreme adhesion and neuronal induction properties, exploring its effect on the adhesion, proliferation, differentiation and other biological behaviors of iNSCs; Thirdly, transplanting iNSC-PLCL/CA-SF in rats model of spinal cord injury to explore whether the tissue engineered spinal cord could promote axonal regeneration across scar interface, form functional neural connections to achieve the purpose of repairing and reconstructing of the injured spinal cord.

脊髓损伤后，轴突虽可再生但不能通过瘢痕界面已成为影响脊髓损伤疗效的关键问题。我们已证实，应用小分子蛋白能够诱导人脐带间充质干细胞分化为神经干细胞，但其效率低、重复性差。许多基因能够促进神经组织的发生、分化和成熟，如过表达Tet3能够更加有效地获得成熟神经元。然而，单纯地移植神经干细胞因缺乏良好支撑，使其修复脊髓损伤的效果欠佳。我们设想，如果能够更加有效地获得神经干细胞并找到一种可靠的支持物供其生长，就可以更好地修复脊髓损伤。本课题首先通过在人脐带间充质干细胞中过表达Tet3诱导其分化为神经干细胞；其次，构建具有良好粘附性及诱导定向生长性能的PLCL/CA-SF三维纳米神经导管，体外研究该导管对iNSC黏附、增殖、分化等生物学行为影响；再次,将iNSC-PLCL/CA-SF植入大鼠脊髓损伤模型，检测该组织工程化脊髓能否促进轴突再生通过瘢痕界面，形成功能性的神经连接，达到修复和重建脊髓的目的。

脊髓损伤(spinal cord injury, SCI)是一种严重的神经系统疾病，预后极差，为患者家庭及社会带来了严重的经济负担。脊髓损伤后，损伤部位的少突胶质细胞大量坏死凋亡，内源性的细胞再生不足以补充组织缺损重新建立连接。许多研究尝试通过细胞给药来填充SCI引起的空洞，促进脊髓再生。因此，建立可靠的外源性人少突胶质前体细胞（oligodendrocyte progenitor cells，OPCs）来源用于细胞移植至关重要。然而，由于资源匮乏和伦理问题，从人类获取同种异体OPCs是困难和不切实际的。人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUMSCs)作为OPCs的潜在来源可能具有优势。5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5hmC）在中枢神经系统中含量丰富。10-11易位蛋白（ten-eleven-translocation protein，TET3）通过调控神经发育相关基因 5hmC的水平，可以影响神经系统的正常发育和功能。我们的研究证明了TET3可以快速有效地将HUMSCs直接转化为OPCs，诱导得到的细胞高表达少突特异性标志物NG2与PDGFRA，伴随着诱导进行，细胞的甲基化活动逐渐活跃，5hmC，去甲基化蛋白TET3、甲基化转移酶1（DNA methyltransferase 1，DNMT1）以及甲基化转移酶3A（DNA methyltransferase 3A,DNMT3A）逐渐上升,提示着细胞内正向神经分化进行重编程。将诱导得到的少突胶质前体细胞作为细胞治疗的种子细胞进行移植可明显增强脊髓损伤后的髓鞘再生。综上所述，TET3可调控HUMSCs向OPCs方向的分化，并有足够的潜力成为细胞移植治疗脊髓损伤的一个重要因素。