【作者】 王春婷；

【导师】 鞠躬；

【作者基本信息】 第四军医大学 ， 神经生物学， 2010， 博士

【摘要】 雪旺细胞(SCs)是周围神经系统的主要胶质细胞,能够合成和分泌细胞表面粘附因子、细胞外基质蛋白及多种神经营养因子和相关受体,以促进轴突再生。雪旺细胞移植已被广泛应用于CNS脊髓损伤修复研究中。联合其他手段移植雪旺细胞能够更好得促进轴突再生和功能恢复。激活态SCs是修复脊髓损伤最佳的种子细胞之一,其分泌的大量的神经营养因子能够改善损伤区的微环境,促进损伤脊髓的功能恢复。体外培养SCs是自体移植的唯一途径,因此获得大量的保持其正常功能的SCs是我们关注的焦点。近年来,许多研究发现锂能够通过多种途径抑制GSK3b,从而启动促进细胞生长的保护机制。锂可以刺激干细胞、纹状体和前脑细胞等多种细胞的增殖,是一种低诱变和低致癌剂。此外我们还注意到胚胎干细胞培养液中白血病抑制因子能够调节细胞的分化、增殖和分型,而其中的2-巯基乙醇也可以促进细胞的增殖并且提高DNA的合成。因此本研究着眼于研究锂剂和胚胎干细胞条件培养液对SCs增殖、存活和功能的影响,并得到如下结果:1.根据不同类型细胞在覆层Lamminin的培养器皿上贴壁速度的差异,我们建立了一种简便、快捷分离纯化成年SCs的有效方法,获得了大量高纯度的自体SCs。纯化培养至3d,5d,大部分双极、三极样细胞胞体发出较长的突起,平行排列成束状。7-10d时基本融合成片。在细胞胞体及突起均有S100表达。培养5-10d,细胞增殖活性逐渐增高。7d时达最高峰,平稳持续至10d缓慢下降,14d后呈明显下滑趋势。流式细胞仪检测到纯化后细胞纯度可高达91%。2. 1mM和5mM氯化锂能够增强体外培养SCs的存活,其中5mM锂浓度作用最强,而0.1mM氯化锂则无此作用,高浓度10mM氯化锂甚至会产生毒性抑制作用,胚胎干细胞培养液培养的SCs与1mM锂作用效果接近,细胞生长状态及速度优于对照组SCs,但弱于5mM氯化锂组。3.相对于对照组而言,在不同时间点,5mM氯化锂组与干细胞条件培养基均对成年大鼠激活态SCs增殖能力有着显著的改善作用。4.通过体外共培养模型,运用免疫荧光标记方法观察锂剂诱导的成年激活态大鼠SCs对胚胎DRGn(dorsal root ganglia neuron背根节神经元)的轴突或突起生长的影响。与对照组相比,接触培养组单个神经元总突起和最长突起的平均长度均显著增长,SCs能够指引神经元突起生长的方向,轴突沿着SCs胞膜及突起伸展的方向迁移并保持密切的接触,提示细胞膜表面粘附分子是支持与引导神经突起生长的重要因素。此外在接触培养组,相较laminin基质层而言,神经元更加偏爱SCs,绝大部分DRGn生长在SCs聚集区内。同样,SCs对中枢海马神经元也有促进突起生长及引导支持的作用。5.在SCs/DRG共培养7d时,SCs突起开始粘附并紧贴DRG神经元突起生长,在此阶段,大部分SCs聚集在DRG神经元附近,胞体呈伸长样,围绕轴突延伸其细长突起。共培养10d后,培养物中开始出现MBP阳性表达,但并非在所有SCs突起上都有表达。随着时间的延长,在14d时,MBP阳性表达开始增强增多,可以看到大部分细胞突起都有表达。此时在电镜下观察SCs可以形成许多质膜包卷或纳入大的轴突。可看见围绕在SCs/轴突复合物的基板的形成。许多分化的SCs形成轴突系膜,松散包绕轴突至少2圈以上,形成幼稚型髓鞘。在SCs/DRG共培养35d时,光镜下观察到SCs包绕的轴突增粗增厚,形成明显可见的黑色条带(Fig.17A、B)。透射电镜下观察其超微结构,可看到紧密型髓鞘形成,约有十几层紧密薄层包绕粗大的轴突,轴突内散布有成束的小圆形微管。有许多细胞SCs以质膜包卷轴突全长,与轴突1∶1形成10-30层的紧密髓鞘板层,即为成熟型髓鞘。提示锂剂预处理的SCs在体外培养中经历了不同的髓鞘形成阶段。综上所述,经laminin选择纯化、锂剂预处理后的SCs在体外培养模型中仍然保持有正常的功能,没有出现功能丧失或缺无。与正常未处理SCs一样,具有支持、引导轴突或突起生长、延伸以及成髓鞘功能。那么这种经锂剂诱导增殖的SCs是安全有效的,它为我们下一步在体观察及移植治疗奠定了坚实的基础。更多还原

【Abstract】 Schwann cells are the myelinating glial cells in the PNS and play a key role in Wallerian degeneration and subsequent regeneration due to their ability to dedifferentiate, migrate, proliferate, express growth promoting factors, and myelinate regenerating axons. In animal models of SCI, grafting Schwann cells or peripheral nerve into the lesion site has been shown to promote axonal regeneration and myelination. Combinatorial strategies with schwann cells and other emerging strategies for spinal cord repair to maximize axonal regeneration and functional recovery.Activated Schwann cell is one of the optimal cell candidates for the repair after SCI. Numerous NTFs secreted by AASCs can improve the microenvironment in the lesion site and promote functional recovery of spinal cord injured rats. Importantly, the development of in vitro systems to harvest human Schwann cells presents a unique opportunity for autologous transplantation in the clinic. To employ Schwann cells for transplantation into the spinal cord, large numbers of cells will be necessary.Recent studies revealed a mechanism that ties together these disparate effects of lithium. Lithium acts through multiple pathways to inhibit glycogen synthetase kinase-3 beta (GSK3b) and turn on cell growth and protection programs. Lithium also stimulates proliferation of stem cells, including bone marrow and neural stem cells in the subventricular zone, striatum, and forebrain. More Importantly, Lithium has low mutagenic and carcinogenic risk. Moreover we also noticed that 2-Mercaptoethanol may induce cell proliferation and enhance DNA synthesis and leukemia inhibitory factor can regulate cell differentiation, proliferation and phenotype. Therefore, we investigate the Effect of lithium and embryonic stem cell culture medium, respectively, on activated Schwann cells proliferation and survival in vitro and got the following results:1. Culture, purification and biological characters of adult Schwann cells from rat predegenerated sciatic nerve sciatic nerves from adult male rat were transected at the midthigh。Seven days later,the distal segment of the transected nerves were removed and used for adult SCs cultures。A simple, inexpensive method for purifying SCs, in which various harvested cell types showed different rates of attachment to the coated laminin culture ware in the initial stage of primary culture, was developed.2. The purified Schwann cells still exhibited a spindle-like shape with bipolar or tripolar morphology at 3 div, 5div, and align themselves parallel to each other in confluent cultures at 7div and 10div. Immunostaining for S100 antibodies showed that the staining was evident in the cell soma and the processes. The proliferation of purified SCs was monitored by visualizing Brdu incorporation using immunocytochemistry. The flow cytometric results which provided quantitative evidence of SCs purity was about 93% at 7 days in vitro. And within 10 day, SCs purity could reach above 91%.3. lower concentrations of lithium 0.1mM did not increase the total number of cells compared to the basal control level. Whereas the total number of cells cultured with both 1mM and 5mM lithium chloride was significantly higher than the control culture. Moreover,Treatment with lithium 10mM sharp decreased the number of cells compared with control,Suggested that high concentrations of lithium may be toxic to SCs. The total number of cells cultured with embryonic stem cell culture medium treatment significantly higher than the control culture.4. Five millimole of lithium maximally stimulated SCs proliferation in culture. Treatment with 5mM Lithium significantly increased the number of BrdU-positive Cells compared with control at both 5 day and 9 day. Likewise, the number of BrdU-positive cells cultured induced by 5mMLiCl cultures or condition medium was significantly higher than the control culture at different time points .5. The ability of these cells to be amplified still retain normal functionality is crucial. We demonstrated that when SCs were allowed to directly contact neurons, SCs could direct the orientation of regrowing neurits, and that axons grew parallel to the long axis of underlying fusiform SCs. In addition, both neuron bodies and axons preferred SCs over artificial substrates in the direct contact coculture.6. To test the ability of the amplified SCs to relate to neuronal surfaces, cells were periodically seeded onto DRG neurite beds and maintained for 7-14 days. To better visualize myelin formed in the cultures, selected cultures were immunostained for the myelin basic protein-MBP. When cells were planted onto neurites, they were seen to line up along the neurites and proliferate. Within the first week, the SCs flattened out on the neurites, extending processes along the axons. Myelin was first observed at 14d. And then, the number of myelin segments increased with time. At the ultrastructural level, some of the differentiated schwann cells formed a mesaxon that spiraled up to 3 turns around an axon at this stage of 14 DIV. Several very thin membranous with no compacted myelin were revealed around an axons. Compact myelin was revealed in co-cultures at 35 DIV. Many cases of segregated large axons were associated 1:1 with SCs and were wrapped with 10-30 layers of compast myelin sheath.From above results we can draw the conclusions that Lithium (1mM, 5mM) and embryonic stem cell culture medium, respectively, could improve significantly activated Schwann cells proliferation in vitro. However, higher concentrations of lithium at 10mM did not increase proliferation. Activated Schwann cells stimulated by lithium could support neurite outgrowth and myelinate axons in vitro.更多还原