【作者】 何继银；

【导师】 劳杰； 赵新； 顾玉东； 虞聪； 张丽银；

【作者基本信息】 复旦大学 ， 外科学， 2007， 博士

【摘要】 前言周围神经的缺损，尤其是长段周围神经的缺损的修复是外科领域的一大难题。随着组织工程材料的不断发展和对于组织工程神经认识的不断深入，使得人工神经的发展成为一个非常有前景的领域。胶元-几丁糖有可能作为人工神经支架的良好材料；激活态雪旺细胞是一种具有高活性、快速增殖的可以显著促进周围神经再生的细胞，是组织工程人工神经良好的种子细胞。本课题将要分三个部分研究为何激活态雪旺细胞有优于正常态雪旺细胞的生物活性?第一部分激活态雪旺细胞信号转导通路的分子构成目的探索激活态雪旺细胞信号转导通路的分子构成。方法用改良成年SD大鼠雪旺细胞培养法培养激活态雪旺细胞(ASC)和正常态雪旺细胞(NSC)，分别用RTK和G蛋白耦联的信号转导通路中的七个抑制剂，Western Blot法检测细胞内的p-ERK1／2水平的改变，比较激活态雪旺细胞和正常态雪旺细胞的信号转导通路的差异。结果光镜下激活态雪旺细胞和正常态的雪旺细胞形态上无差异，S-100染色均为阳性，Western Blot法检测ASC和NSC细胞内的ERK1／2水平无明显的差异，但ASC内p-ERK1／2水平有显著差异(p＜0.01)。在激活态雪旺细胞，使用抑制剂CTX、D609和BAPTA-AM细胞内的p-ERK1／2水平显著降低，而其它抑制剂使用后细胞内的p-ERK1／2水平无明显的改变，差异具有显著性(p＜0.01)；在正常态的雪旺细胞，使用抑制剂U73122和BAPTA-AM，细胞内的p-ERK1／2水平显著降低，而其它抑制剂使用后细胞内的p-ERK1／2水平无明显的改变，差异具有显著性(p＜0.01)。结论激活态雪旺细胞和正常态雪旺细胞是同一细胞的二种不同存在的状态，有活性的p-ERK1／2的水平的升高可能是雪旺细胞激活的关键环节。激活态雪旺细胞和正常态雪旺细胞都是通过G蛋白耦联受体信号转导通路来发挥生物效应的，但是激活态雪旺细胞通过PC-PLC分解磷脂酰胆碱使细胞内的DAG水平的提高来发挥生物效应的，而正常态雪旺细胞则通过PI-PLC信号转导通路发挥其生物效应的。第二部分应用转录因子蛋白／DNA组合芯片技术检测激活态雪旺细胞的活性转录因子目的检测激活态雪旺细胞活性转录因子。方法应用转录因子蛋白／DNA组合芯片技术检测激活态雪旺细胞的活性转录因子。结果激活态雪旺细胞和正常态雪旺细胞的转录因子芯片点阵图显示：激活态雪旺细胞比正常态雪旺细胞有5个转录因子蛋白上调2倍以上，它们是AP-1、NRF-1、NF-Atx、PO-B和PRDI-BF；活化的AP-1蛋白的显著增加可能就是激活态雪旺细胞增殖活性高重要的原因；NF-Atx表达升高可能是协同AP-1的转录表达；激活态雪旺细胞可能通过提高NRF1和mtTFA表达，引起线粒体呼吸链酶蛋白表达升高，线粒体酶活性升高，从而提高线粒体能量代谢，为激活态雪旺细胞的高分泌状态提供能量，促进激活态雪旺细胞的增殖；而PO-G不仅作用于它的靶基因，而且还作用于其它的基因位点，调节转录和DNA的修复和复制。PRDI-BF的升高也与激活态雪旺细胞的特殊的功能密切相关的。结论5个转录因子蛋白上调都与激活态雪旺细胞的快速分裂、高分泌状态密切相关。第三部分应用双荧光素酶报告基因检测系统检测激活态雪旺细胞信号转导通路目的应用双荧光素酶报告基因检测系统检测激活态雪旺细胞信号转导通路。方法采用荧火虫荧光素酶和海肾荧光素酶组成双荧光素酶报告基因实验系统，构建含NGF-TF与promotor／TRE的启动子的载体，感染激活态雪旺细胞，雪旺细胞的处理则分为7组：1．激活态雪旺细胞空白对照：2．激活态雪旺细胞+0.8μg pGL3+40 ng phRL-SV40；3．激活态雪旺细胞+0.8μg pGL3-NGFpromotor+40 ng phRL-SV40；4．激活态雪旺细胞+0.8μg pGL3-NGFpromotor+40 ngphRL-SV40+加药0.5小时；5．激活态雪旺细胞+0.8μg pGL3-NGFpromotor+40 ng phRL-SV40+加药1小时；6．激活态雪旺细胞+0.8μg pGL3-NGFpromotor+40 ng phRL-SV40+加药1.5小时：7．激活态雪旺细胞+0.8μg pGL3-NGFpromotor+40 ng phRL-SV40+加药2小时。结果从第三组加入活化的NGF的启动子但没有加入抑制剂后，RLU1的数值含有激活态雪旺细胞自身的荧光、海肾荧光素酶的荧光强度和萤火虫荧光素酶作用于底物的荧光强度的总和，因而数值最大，其相对值RLU1／RLU2也是最大，使用信号转导抑制剂D609后，第四组在用药0.5小时后，由于NGF启动子的活化受到抑制，萤火虫荧光素酶作用于底物的强度明显受到限制，表现在荧光强度显著下降。当D609随着时间的推移，逐渐衰减的时候，NGF启动子的活化受到抑制逐渐减弱，萤火虫荧光素酶作用于底物的强度明显增强，表现出荧光强度的逐渐增强。结论通过双荧光素酶报告基因系统可以直观地观察激活态雪旺细胞的信号转导通路的作用效应；直接证明了前面我们的信号转导通路的分子构成的检测结果的正确性。更多还原

【Abstract】 IntroductionIt is a difficult problem in peripheral surgery when nerve deficiency especially long distant nerve deficiency meets. The artificial nerve is now one of prospective areas with the development of biomaterials and deeper study of tissue engineer nerve. Collagen-Chitosan might be an ideal material for fabrication artificial nerve and activated Schwann cell might be the ideal seed cell for the bio-active nerve because of its high bio-active and quick proliferation. In our three part of this research we will study the reason why activated Schwann cell perior nomal Schwann cell to boosting the regeneration of injuied nerve.Part 1: To explore molecular composition of activated Schwann cells’ signal transduction pathwayObjective To explore molecular composition of activated Schwann cells’ signal transduction pathway. Methods Activated Schwann cells and normal Schwann cells were abtained by the way of modified Schwann cell from adult SD rats. 7 signal transduction pathway inhibitors of RTK and GPCR were added into activated Schwann cells and normal Schwann cells for an hour before these cells were collected respectively. Western Blot method was used to check the change of p-ERK1/2 to compare the difference between the activated Schwann cell and normal Schwann cell. Results Both of them were positive stained by S-100．ERK1/2 and p-ERK were also compared in activated Schwann cells and normal Schwann cells. There was no statistic difference of ERK1/2 between the two cells. However p-ERK was remarkable different （p<0．01） . The rising of p-ERK1/2 might be the key reason of Schwann cells activated. In activated Schwann cells, changes of p-ERK1/2 by CTX、D609 and B?PTA-AM were prominent but there was no distinct change using other inhibitors （p<0．01） . However in normal Schwann cells, changes of p-ERK1/2 inhibitor U73122 and BAPTA-AM were notably but there was no distinct change using other inhibitors （p<0．01） . Conclusions Activated Schwann cell and normal Schwann cells is the same cell in two different conditions. The rising of p-ERK1/2 might be the key reason of Schwann cells activated. GPCR was the common signal transduction pathway of Activated Schwann cell and normal Schwann cells. But Activated Schwann cells exert their biologic effect through PC-PLC decompounding choline and boosting level of DAG. However normal Schwann cells exert their biologic effect through PI-PLC signal transduction pathway.Part 2: To explore the activated transcription factors of activated Schwann cell using TranSignal^TM Protein/DNA Array techniquesObjective To explore the activated transcription factors of activated Schwann cell. Methods TranSignal^TM Protein/DNA Array techniques were adopted to test the activated transcription factors. Results Lattice of activated Schwann cells showed that there were 5 different transcription factors doubly raised their expression than that of normal Schwann cells. The rising of AP-1 might be the dominant reason of activated Schwann cells’ quick proliferation. NF-Atx takes a cooperation role with AP-1．NRF1, however, may supply the activated Schwann cells with energy with its enzyme mtTFA for its’ quick proliferation and vigorous secretion. PO-G exerts its action in DNA repair and replication of activated Schwann cells. Finnally PRDI-BF ties closely up with the special condition of activated Schwann cells. Conclusion Total 5 different transcription factors correlated their level with activated Schwann cells’ quick proliferation and vigorous secretion condition .Part 3: To examine the signal transduction passway of activated Schwann cell using the method of dual color fluorescence reporter gene system.Objective To examine the signal transduction of activated Schwann cell using the method of dual color fluorescence reporter gene system. Methods Dual color fluorescence reporter gene system made of pGL3 Luciferase Reporter and phRL-SV40 was adopted and vector contained NGF-promotor.Activated Schwann cells were infected by these vectors. After that Schwann cells were divided into 7 teams. They were: 1．Activated Schwann Cell（ASC）, 2．ASC+ 0．8μg pGL3 + 40 ng phRL-SV40, 3．ASC+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 4．ASC+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 + add D609 0．5h, 5．ASC+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 + add D609 1h,6．ASC+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 + add D609 1．5h, 7．Cell+ 0．8μg pGL3-NGFpromotor + 40 ng phRL-SV40 + add D609 2h. Results Fluorescence of 3th team was the most intensive in the 7 teams. Its comparative value RLU1/RLU2 was also the biggest. However 0．5h after the usage of signal transduction inhibitor D609 the value of ASC decreased suddenly due to the activating of NGF-promotor was restrained. And pGL3 luciferase could not call into play. But alone with the attenuation of D609, the activated level NGF-promotor increase gradually. And pGL3 luciferase resumed their role playing on their substrate and the fluorescence intensity was also increased gradually. Concision The effect of signal transduction of activated Schwann cells on their target were observed directly through Dual color fluorescence reporter gene system. And our molecule composition of signal transduction pathway of activated Schwann cell were proved right directly.更多还原