S100β基因修饰的嗅鞘细胞联合异种神经桥接体修复坐骨神经损伤的机制

Peripheral nerve injury (PNI) is common and resulting in considerable long-term disability，but the clinical outcome is often not optimal. Although some positive effect was get by combining the bridged material of no antigen with olfactory ensheathing cells (OECs) transplantation to treatment the PNI, the method was limited by the transplanted OECs' survival number under the inflammation condition, which would lead to the poor repairing effect. S100β plays the important role in cell proliferation and maintaining the axonal structure of neuron, it may also decrease some proinflammation factor, but its detail molecular function is still unclear. We have found the low expression level of S100β in the transplanted part, when utilized the bridged material of no antigen with olfactory ensheathing cells to repair the sciatic nerve injury at the acute inflammation phase. In our study, we want to check whether the S100β gene modified OECs combining with the bridged material of no antigen can promote the survival of donor cell (OECs) and the function of the host neuron, and then we will clarify the underlie mechanism. The molecular role of S100β in the cellular survival ability and the spinal ganglia cell axonal extendedness will be checked with an established in vitro inflammation model. The viability of OECs will be measured by MTT; the apoptosis will be evaluated by TUNEL staining and gene expression of apoptosis related factor; the expression of proinflammation related factor and S100β will be checked by real time PCR and ELISA; the axonal proliferation related protein will be check by immunocytostaining. Then the involvement of S100β in the NFκB/MAPK signaling pathway will be analyzed. The transcriptional factors will be check by western blot. The repairing effect of the S100β gene modified OECs combining with the bridged material of no antigen will be evaluated after grafting them to the rat sciatic nerve injury model by measuring the recovery of function, and then the underlies mechanism will be analyzed. What our finding in present study will be provide the possible method and fundamental theory for clinical effective treatment to PNI.

周围神经损伤后急性期，损伤部位炎性反应导致移植的嗅鞘细胞（OECs）存活率低可影响神经损伤修复效果。S100β对提高神经细胞的生存能力及维持神经元轴突的结构有重要作用。我们前期实验发现，周围神经损伤急性期进行桥接体移植后，移植桥接体区及远侧端神经段S100β表达水平低下，影响了OECs存活率。本项目构建S100β基因修饰的OECs培养体系（S100β(+)OECs）和S100β(+)OECs/神经节细胞共培养体系，并建立炎症模型，证明S100β(+)OECs在炎症环境下的生存能力及其对周围神经元的影响，阐明S100β通过NFκB/MAPK及RAGE信号通路调控神经细胞增殖的机制；将S100β(+)OECs联合异种神经桥接体移植于大鼠坐骨神经缺损模型，验证S100β(+)OECs存活状态和生物活性，探讨其对周围神经损伤后炎症期的修复效果及其机制，为临床周围神经损伤治疗提供新方向。

本项目针对在周围神经急性损伤期内移植嗅鞘细胞（olfactory ensheathing cells，OECs）进行神经修复治疗时，OECs存活能力低，修复效果不理想等问题，基于“S100β可以延长OECs的生存能力，并有助于OECs促进外周神经元轴突功能恢复”的实验假设，利用体外培养模型探讨了S100β对OECs的生存能力及其对周围神经元轴突生长的影响。为了明确S100β对重组干扰素-γ（Interferon-γ, IFN-γ）诱导的炎症条件下OECs的作用及其机制以及S100β联合OECs对炎症条件下周围神经元轴突修复功能的影响。实验将采用改良差速贴壁联合胰酶限时消化3 min法纯化培养的大鼠OECs或脊神经节细胞（dorsal root ganglia cell, DRG）分为空白对照组、炎症模型组、S100β组等3组，每组种板1×106/ml；空白对照组采用常规培养体系培养细胞（OEC或DRG），炎症模型组采用IFN-γ诱导炎症建立细胞体外炎症模型，S100β组在炎症模型组基础上加入S100β蛋白进行干预；采用免疫荧光染色进行细胞鉴定、纯度分析及凋亡检测；利用MTT法检测细胞的增殖情况并筛选IFN-γ诱导细胞炎症模型的适宜浓度；利用TUNEL染色法判断各组细胞凋亡状况；采用Real-Time PCR检测各组细胞表达凋亡相关因子Bcl 2，Bax，Puma及炎症相关因子iNOS、IL-1β、TNF-α的mRNA水平。根据以上结果，判断在炎症环境中S100β对细胞的影响及可能机制。结果发现：采用改良方法原代培养的大鼠OECs及DRG均能够获得80%纯度。与5 ng/ml的浓度相比，10 ng/ml的IFN-γ对OECs的抑制率更高，凋亡细胞更多。DRG细胞增殖能力变化与IFN-γ浓度呈浓度依赖性关系。与炎症模型组比较，S100β干预组细胞增殖能力升高，凋亡细胞减少；S100β干预组细胞炎症相关因子iNOS、IL-1β以及TNF-α的基因表达水平下降。以上实验结果表明：S100β可以降低细胞凋亡发生，下调IFN-γ激活的炎症因子iNOS、IL-1β和TNF-α的基因表达水平。提示S100β可能是通过抑制炎症因子的表达从而减少了细胞凋亡的发生。本研究结果为深入研究S100β联合嗅鞘细胞移植对周围神经损伤的修复作用奠定了基础。