失神经Schwann细胞在干细胞移植修复陈旧性周围神经损伤中的作用

Stem cell transplantation has been investigated as a promising treatment strategy for peripheral nerve injury. Recent studies have shown that the transplantation of stem cells of different origins has favorable effects on peripheral nerve regeneration. The mechanism underlying the beneficial effect of implanted stem cells from various sources on peripheral nerve regeneration remain unclear. Although the cell replacement, trophic factor production, extracellular matrix molecule synthesis, guidance, remyelination, microenvironmenral stabilization, and immune modulation have been postulated as possible mechanisms for stem cell implantation. It is still largely unknown.We wonder what happen to denervated schwann cell after transplantation of stem cells. Be replaced or re-activated by implantated stem cells? Why stem cells derived neurons can survive in peripheral nerve? What does it mean? It remain unclear that the capability of long-term denervated schwann cells(SCs) to promote axonal regeneration in the field of cellular therapy in peripheral nerve regeneration so far. Our hypothesis is that implantated stem cell-derived neutons through axonal contact signal or neurotrophic function to save the denervated SCs from apoptosis, or trigger SCs proliferation. Axonal contact re-established in a timely fashion make bund of Bungner retain and the integrity of the basement membrane, and promote the nerve regeneration. To test this hypothesis, cross anastomosis model of rabbit after various periods of denervation were used by suturing the proximal stump of freshly transected common peroneal nerve end-to-end to the denervated distal stump of the tibial nerve.The re-activation of denervated SCs will be assessed after transplantation of neural tissue-committed stem cells (NTCSCs)in bone marrow、or neurons、schwann cells derived from NTCSCs.To determinate whether it is by axonal contact or neurotrphic function,the neurotrophic factors such as GDNF and neuregulins also be used for comparison. The mechanism of stem cell transplantation to repair peripheral nerve damage will be more clarified by this study. In addition to replace the cells, it is the more important that stem cells derived neurons may provide the axonal contact to re-activate denervated schwann cells which maintain its function and viability to promote nerve regeneration and promote nerve functional recovery.

干细胞研究为周围神经损伤后神经功能修复提供了广阔应用前景。但其机制不清，目前研究主要集中在细胞移植后存活、分化及神经通路的建立，却忽略了宿主失神经Schwann 细胞的作用。有研究显示神经元能在周围神经存活，且形成神经-肌接头。我们前期实验显示干细胞源性神经元构建的组织工程神经移植促进大鼠及犬坐骨神经功能恢复。但干细胞源性神经元在周围神经存活的意义是什么？其促进神经功能修复的机制是什么？我们推想植入的干细胞源性神经元通过神经营养或细胞接触信号作用支持或活化宿主失神经Schwann细胞，维持Bungner带和基膜完整性，从而促进神经再生。为验证这一假说，我们拟采用兔陈旧性胫神经横断性损伤远侧端与新鲜腓总神经横断近侧端交叉吻合模型，研究骨髓NTCSCs源神经元对宿主失神经Schwann细胞的影响，这对深入理解干细胞源性神经元、以及干细胞移植修复周围神经损伤机制具有重要的理论及应用价值。

周围神经损伤不能获得理想功能恢复主要的原因是随着损伤时间的延长，神经损伤远侧段失神经Schwann细胞支持神经再生的能力逐步减弱，故本课题拟应用干细胞源性神经元移植活化或减少schwann细胞凋亡，从而促进神经再生及神经功能恢复。. 体外实验：（1）大鼠骨髓神经组织定向干细胞的分离、培养及鉴定；（2）应用小分子化合物将NTCSCs或BMSCs诱导成神经元，应用免疫荧光、RT-PCR和Western blot进行鉴定；（3）分离、培养大鼠坐骨神经损伤远侧段失神经Schwann细胞，构建大鼠骨髓源性神经元与失神经Schwann细胞体外共培养体系，检测骨髓源性神经元对Schwann细胞增殖的作用，检测骨髓源性神经元对Schwann细胞神经营养因子和粘附分子的分泌功能。（4）骨髓源性神经元与损伤神经远侧段体外共培养，检测骨髓源性神经元对损伤神经远侧段Schwann细胞的增殖作用。. 体内实验：构建大鼠坐骨神经鉗夹性损伤模型（1）检测大鼠骨髓源性神经元样细胞在体内的存活；（2）检测植入骨髓源性神经元细胞对Schwann细胞形态和数量的影响；（3）检测骨髓源性神经元细胞对Schwann细胞增殖的作用；（4）检测骨髓源性神经元样细胞对Schwann细胞凋亡的作用。（5）检测骨髓源性神经元样细胞对轴突形态和数量的影响；（6）检测骨髓源性神经元样细胞对髓鞘再生的作用；（7）检测骨髓源性神经元样细胞对神经功能恢复的作用。. 结果：（1）骨髓源性神经元细胞促进Schwann 细胞增殖；（2）骨髓源性神经元细胞增强Schwann 细胞分泌神经营养因子和粘附分子能力；（3）骨髓源性神经元细胞减少Schwann细胞凋亡；(4 )植入体内的骨髓源性神经元细胞与周围schwann细胞形成紧密接触，再生轴突明显增加，Schwann细胞的再髓鞘化数量增多，髓鞘板层保持致密。足迹分析显示骨髓源性神经元样细胞植入促进坐骨神经功能恢复著。. 结论: 体内外实验结果提示骨髓源性神经元细胞抑制Schwann细胞凋亡，促进其增殖，以及促进其分泌神经营养因子和粘附分子，促进周围神经再生、再髓鞘化及神经功能恢复，成功验证本课题提出的科学假说，为临床干细胞源性神经元移植提供了理论基础。