【作者】 刘欣春；

【导师】 朱悦；

【作者基本信息】 中国医科大学 ， 外科学， 2007， 博士

【摘要】 前言中枢神经系统自我修复能力有限，损伤的神经元不能再生，细胞替代疗法是其治疗策略之一。神经干细胞因具有连续增殖能力及神经世系的分化潜能而成为中枢神经系统损伤替代疗法的候选细胞之一。基于神经干细胞的修复策略有两类，一是通过调控体内神经干细胞的增殖分化来实现，二是通过神经干细胞移植替代丢失的神经组织。本课题是关于神经干细胞分离培养及分化方面的研究。立题一：低温保存流产儿脊髓神经干细胞的分离培养为了比较不同来源神经干细胞的特性，探寻适合移植治疗的神经干细胞来源，人们已经从除成人脊髓外的人胚和成人各个脑区分离神经干细胞进行研究，这些人体神经组织大部分都是新鲜取材立即培养，少部分取材后经过营养液中的短暂保存后培养，然而工作中常遇到不能立即取材的情况，因此本立题将14周龄流产儿不经任何处理置于冰箱中低温保存一段时间后，分段进行脊髓神经干细胞的分离培养，检测能否分离培养出神经干细胞，并对分离的干细胞的克隆形成能力、分化潜能等性质进行进一步分析比较。立题二：新生小鼠端脑多能神经前体细胞分化成运动神经元的研究神经干细胞移植到宿主中枢神经系统后的迁移分化情况在很大程度上受移植处微环境的影响，损伤后的中枢神经系统则对神经干细胞的分化更具选择性作用，植入的神经干细胞大多分化成胶质细胞，这也是利用神经干细胞进行神经修复需要解决的问题。为了使移植的细胞如期分化为神经元细胞，人们尝试了神经元前体细胞移植，虽然神经元前体细胞在移植后不分化成胶质细胞，但在损伤区的成熟受到限制，即表达特异类型的神经递质受到限制。因此在移植前把干细胞诱导分化成特定类型的神经元更具可行性。运动神经元因其在神经疾病中的重要性及发育机制相对清楚，是目前诱导分化研究较多的神经元类型。运动神经元已经证明能够从胚胎干细胞及胚胎神经干细胞诱导分化，本立题探寻新生期小鼠端脑多能神经前体细胞是否能够分化成运动神经元，并探寻提高其分化效率的方法，为运动神经元诱导分化提供新的细胞来源。立题三：OMgp对神经干细胞分化的影响神经干细胞植入成体非神经发生部位或损伤区绝大部分分化成胶质细胞，但植入发育期神经组织中能够分化成神经元，目前还不清楚造成这种差异的原因。发育期中枢神经系统与成熟中枢神经系统的区别之一在于成体有发育完好的髓鞘，而且髓鞘是脊髓损伤后抑制轴突再生的主要局部环境因素之一，那么，前述神经干细胞移植后的分化现象是否也与髓鞘有关呢?因此，本立题探讨髓鞘的主要活性成份之一OMgp(oligodendrocyte-myelin glycoprotein)对神经干细胞分化是否具有影响，同时探讨OMgp对干细胞源性神经元轴突生长是否也有的影响，并检测NGF、dbcAMP(dibutyryl cAMP)对OMgp是否具有对抗作用。材料与方法立题一：14周流产儿在4℃下保存，于2h、6 h和12h后取脊髓，颈段、胸段、腰骶段分别进行无血清培养，并用胎牛血清诱导分化。用克隆培养的方法验证培养细胞的干细胞特性。用免疫荧光细胞化学的方法检测神经干细胞标志nestin及干细胞诱导分化后神经元标志MAP2、星形胶质细胞标志GFAP、胆碱能标志ChAT，比较不同时间点以及不同部位分离神经干细胞的差异。立题二：取新生小鼠端脑进行多能神经前体细胞的分离培养，用无血清培养基培养，8-10天后用5％胎牛血清诱导分化，并用RA(retinoic acid)、Shh(sonic hedgehog)以及dbcAMP来提高运动神经元的分化效率，利用免疫荧光的方法检测运动神经元标志物MAP2、ChAT及HB9来证明运动神经元的分化；利用神经前体细胞与骨骼肌共同培养，通过免疫荧光方法检测MAP2、N-AChR共存来证明分化运动神经元与肌细胞能够形成突触结构，并运用药理学方法证明突触能够传递冲动。立题三：取新生小鼠端脑进行神经干细胞的分离培养，用5％胎牛血清诱导分化，并将其分为四组：将分化培养分成4个组，组1为阴性对照，组2加入终浓度为50 ng／ml OMgp，组3加入终浓度为50 ng／ml OMgp+100 ng／ml NGF，组4加入终浓度为50 ng／ml OMgp+1000μM dbcAMP，培养4天后用免疫荧光的方法检测神经干细胞分化后神经元标志MAP2、星形胶质细胞标志GFAP，并计算并比较各组神经元与胶质细胞的比例，测量并比较各组神经元轴突的长度。实验结果立题一：在各个时间点上，颈段、胸段、腰骶段脊髓均分离培养出具有连续增殖能力的神经球，腰骶段分离出的神经球数量最多，12 h组各段分离出的神经球较2 h、6 h组显著减少。各培养中神经球均为nestin阳性，诱导分化后均能够产生GFAP阳性星形胶质细胞、MAP2阳性神经元以及ChAT阳性的胆碱能神经元。各培养中神经干细胞的克隆形成能力相似。立题二：从新生小鼠端脑成功分离培养多能神经前体细胞，具有连续增殖能力，能分化形成MAP2阳性神经元以及GFAP阳性星形胶质细胞，4％的分化细胞为ChAT~+的胆碱能神经元，1％的细胞表达运动神经元特异性标志物HB9，当加入运动神经元诱导因子后，HB9~+细胞数增加到5％。神经前体细胞与骨骼肌细胞共培养时可见分化的运动神经元与骨骼肌形成突触结构，并能够传导冲动。立题三：神经干细胞正常分化培养中MAP2阳性神经元比例13.85％，GFAP阳性星形胶质细胞比例3.15％，加入OMgp后神经元比例降至3.40％，星形胶质细胞比例升至6.01％，同时轴突生长受抑；NGF和dbcAMP对OMgp有拮抗作用。讨论立题一：克隆培养证明本研究中分离的细胞能够自我更新、扩增，能够分化成中枢神经系统的主要细胞，符合干细胞的评定标准。腰骶段能够分离更多的神经球说明腰骶段有更多有反应能力的干细胞或腰骶段的干细胞有更强的存活能力。随流产儿体外保存时间的延长，分离的神经球数量减少，说明干细胞死亡逐渐增加。脊髓各节段分离的细胞具有相似的克隆形成能力及分化能力，本质上可认为是同种细胞。立题二：从新生小鼠端脑分离细胞具有连续增殖能力，能分化形成神经元以及星形胶质细胞符合多能神经前体细胞评定标准。部分分化细胞表达运动神经元特异性分子标志HB9说明分化培养中有运动神经元的产生。分化的运动神经元能与骨骼肌细胞形成突触并传导冲动，说明这些运动神经元具有功能性。立题三：OMgp促神经干细胞向星形胶质细胞方向分化，抑制神经元方向的分化；NGF对其有对抗作用；dbcAMP也具对抗作用提示OMgp发挥作用可能通过NgR通路。结论立题一：从低温保存的流产儿能够分离培养出脊髓神经干细胞；腰骶段脊髓分离得到原代神经球最多；原代神经球的数量随着保存时间的延长而减少；低温保存不影响神经干细胞的性质。立题二：新生小鼠端脑多能神经前体细胞能够诱导分化成功能性运动神经元，诱导因子的加入可提高运动神经元的分化比例。立题三：OMgp促神经干细胞向星形胶质细胞方向分化，抑制神经元方向的分化；NGF、dbcAMP具对抗作用。更多还原

【Abstract】 IntroductionCentral nervous system (CNS) has limited ability to repair itself and the lost neurons can not be regenerated. Cell replacement therapy is one of the strategies to repair the damaged CNS. Neural stem cells (NSCs) represent one of the candidate cell types for cell replacement because they have the ability to proliferate continuously in vitro and can differentiate into multiple neural lineage. Strategies based on NSC fall into two categories: stimulating the endogenesis NSC to proliferate and differentiate or transplantation of in vitro expanded NSC to the injury site. The present dissertation includes series of studies on the isolation and differentiation of NSC.Study 1: Isolation of neural stem cells from the spinal cords of low temperature preserved abortusesIn order to find the right source for transplantation, neural stem cells have been isolated from various regions of human fetal and adult brain except for human adult spinal cord. The neural tissues were obtained freshly used for cell culture immediately in most of the studies or after short preservation in culture medium. However, we usually failed to obtain fresh tissues for some delays. Therefore we explore the possibility to isolate spinal NSCs from 14 weeks old abortuses after the preservation in a refrigerator for postmortem intervals. The clonal ability and differentiation potential of the isolated stem cells were analyzed.Study 2: Motor neurons differentiating from neonatal mouse multipotent telencephalon precursor cellsSignals in the local environment play an important role in determining the fate of the neural stem cells after the transplantation to CNS. Neural stem cells are more restricted in damaged CNS and differentiate mainly into glia rather than neuronal cells. These problems need to be solved before the application of neural stem cells for CNS repair. In order to obtain the desired neuronal types, neuronal restricted precursors were tried for transplantation. The precursors failed to mature into neurons expressing neural transmitters although they did not differentiate into glia. Hence, it seems necessary to induce NSCs to differentiate into desired neuronal types. Motor neurons represent one CNS neuronal subtype for which pathways of neuronal specification have been defined and motor neuron differentiation is a hot topic at present. Recently, many studies have obtained motor neurons differentiating from in vitro cultured cells including embryonic stem cells and embryonic neural stem cells. The present study aims at exploring the possibility of motor differentiation from neonatal mouse multipotent telencephalon precursor cells, searching new methods to elevate the differentiation efficiency and providing a new cell source for motor neuron differentiation study.Study 3: The effect of OMgp on neural stem cell differentiationNeural stem cells differentiated mainly into glia rather than neuronal cells when they were transplanted into non-neurogenic or damaged adult CNS. But they could differentiate into neurons when they were implanted into developmental CNS. There is not a convincing explanation for the phenomenon. The matured CNS has well developmental myelin sheath comparing with developing CNS. We doubt that myelin sheath may affect negatively the transplanted NSCs to differentiate into neurons since it has been documented as an axon growth inhibitor. The present study explore whether of not OMgp (oligodendrocyte-myelin glycoprotein) which is main component of myelin sheath has an effect on NSCs differentiation as well on stem cell derived neuronal axon growth. We also test if the effect of OMgp can be reversed by NGF or dbcAMP (dibutyryl cAMP). Materials and MethodsStudy 1: Fourteen weeks old abortuses were stored in a refrigerator at 4℃for 2 h, 6 h and 12 h before use. Neural stem cells were isolated from cervical cord, thoracic cord and lumbar/sacral cord separately and induced to differentiate with fetal bovine serum. Clonal culture was carried out to demonstrate that the isolated cells met the standard of stem cells. Fluorescent immunocytochemistry was used to examine the expression of neural stem cell marker (nestin), neuronal marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures were compared.Study 2: Multipotent neural precursor spheres were isolated and expanded from neonatal mouse telencephalon in serum free medium. They were induced to differentiate by using 5% fetal bovine serum after 8-10 d culture and RA (retinoic acid), Shh (sonic hedgehog) and dbcAMP were employed to increase the efficiency of motor neuron differentiation. Motor neuron marker MAP2, ChAT and HB9 were detected by immunofluorescence. Immunofluorescence colocalization of MAP2 and N-AChR in precursor cell and myoblast coculture demonstrate the formation of motor endplate. Pharmacological approaches were used to prove that the synapse could transmit excitement.Study 3: Neural stem cells were isolated and expanded from neonatal mouse telencephalon. 5% fetal bovine serum was used to induce stem cell differentiation. The differentiation cultures were grouped as follows: group 1 as negative control, group 2 supplemented with 50 ng/ml OMgp, group 3 supplemented with 50 ng/ml OMgp +100 ng/ml NGF, group 4 supplemented with 50 ng/ml OMgp + 1000μM dbcAMP. Fluorescent immunocytochemistry was used to examine the expression of neuronal marker MAP2 and astrocyte marker GFAP. The percentage of neurons and glia and the length of neuronal axons in different groups were compared. ResultsStudy 1: Neural stem cells were obtained from all the spinal cord segments with different postmortem intervals. The lumbar/sacral cord cultures gave rise to the most abundant primary neurospheres. When the preservation was prolonged to 12 h, the number of primary neurospheres decreased sharply. Neurospheres in all cultures showed nestin positive immunoreactivity and could yield GFAP positive astrocytes and MAP2 positive neurons including ChAT positive cholinergic neurons in differential cultures. The clonal formation and phenotype capacity were similar in all cultures.Study 2: Multipotent neural precursor spheres were isolated from neonatal mouse telencephalon and could proliferate continuously. They could differentiate into MAP2~+ neurons and GFAP~+ astrocytes. 4% of the differentiated cells were ChAT~+ cholinergic neurons. 1% of the differentiated cell expressed specified motor neuron marker HB9. The percentage increased to 5% when the inducing factors were added in. The differentiated motor neurons could develop neuromuscular junctions when cocultured with myoblasts and the synapse had the ability to transmit excitements.Study 3: The ratio of MAP2 positive neurons decreased from 13.85% to 3.40% and the ratio of GFAP positive astrocyte increased from 3.15% to 6.01% when OMgp was added into the differentiation cultures. The growth of neuronal axons was also depressed by OMgp. The effect of OMgp could be reversed by NGF or dbcAMP.DiscussionStudy 1: Clonal culture demonstrated the isolated cells in the present study were genuine neural stem cells as they were self-renewal, expandable and could differentiate into the main cell types in the CNS. The fact that more primary neurospheres were obtained from sacral/lumber segment indicates that more responsive stem cells exist in this segment or survived the preservation. The number of neurospheres decreased as the preservation period was prolonged, which indicates that more stem cell died. The cells obtained from all the spinal cord segments may be the same cells in essence as they had very similar clonal and differentiation capacity.Study 2: The isolated and expanded cells met the standards of multipotent neural precursor because they could proliferate continuously and differentiate into neurons and astrocytes. Some of the differentiated cells expressed specified motor neuron marker HB9, which indicated motor neuron differentiation. The differentiated motor neurons were functional because they could develop neuromuscular junctions when cocultured with myoblasts and the synapse had the ability to transmit excitements.Study 3: OMgp had an effect on neural stem cell differentiation by increasing astrocyte lineage differentiation while depressing neuron lineage differentiation. NGF could reverse the OMgp effect. The fact that dbcAMP had antagonism action similar to NGF indicated that NgR might be involved in the pathway.ConclusionsStudy 1: Spinal neural stem cells can be isolated from low temperature preserved abortuses. Sacral/lumber segments give rise to the most abundant stem cells and the number of neurospheres decreases as preservation time goes on.Study 2: Neonatal mouse multipotent telencephalon precursor cells can give rise to functional motor neurons. Inducing factors can elevate the efficiency of motor neuron differentiation.Study 3: OMgp has an effect on neural stem cell differentiation by increasing astrocyte lineage differentiation while depressing neuron lineage differentiation. NGF or dbcAMP could reverse the OMgp effect.更多还原