三七总皂甙对去皮层血管成年大鼠前脑神经干细胞及神经营养因子作用的实验研究

【作者】 程龙；

【导师】 朱培纯；

【作者基本信息】 北京中医药大学 ， 中西医结合基础·神经解剖学， 2003， 博士

【摘要】 近年来大量研究证实，成年哺乳动物脑内仍保留着具有自我更新和多向分化潜能的神经干细胞，它们位于侧脑室的室管膜下层(subependymal ventrical zone, SVZ)和海马齿状回的颗粒细胞下层(subgranular zone, SGZ)。神经营养因子如bFGF、BDNF能促进神经干细胞的增殖、迁移和分化，还具有神经保护与修复的作用,本研究拟探讨去大脑皮层血管对成年大鼠前脑神经干细胞及bFGF、BDNF表达的影响，并试图确定三七总皂甙的治疗作用，拓宽中医祛瘀生新法在脑病治疗中的现代医学理论基础。1 去大脑皮层血管对成年大鼠侧脑室管膜下层神经干细胞影响及三七总皂甙的作用本研究采用Wistar成年大鼠，去除大鼠左侧大脑皮层血管，腹腔注射BrdU标记增殖细胞，应用免疫组化方法观察去皮层血管对SVZ神经干细胞增殖、迁移、分化和bFGF表达的影响，以及三七总皂甙的作用。通过连续切片的免疫组化结果显示去皮层血管后侧脑室从前至后包括前角、中央部和下角SVZ的BrdU、PCNA、Nestin、Tuj-1、Vimentin阳性细胞均明显增多，反应增强，表象改变显著，其中Tuj-1主要在侧脑室前角表达，而在下角主要是胶质细胞表达明显。bFGF在侧脑室各水平的SVZ表达皆增多、增强，提示去皮层血管引起内源性bFGF表达增高，bFGF可能通过自分泌或旁分泌作用于其受体，刺激相对静止的SVZ神经干细胞增殖、分化，给予三七总皂甙后促进以上各种阳性细胞表达增多、增强，提示三七总皂甙可能通过上调内源性bFGF的表达促进去皮层血管后成年大鼠SVZ神经干细胞增殖、分化。2成年大鼠去皮层血管引起室管膜下层细胞增殖形成迁移流至损伤部位本实验观察到：成年大鼠去皮层血管后不仅促进侧脑室前角SVZ细胞及RMS增殖，也引起下角SVZ增殖并形成两条迁移流经胼胝体到达皮质损伤区，其中H链起自下角上壁；L链起自下角外侧壁，来自RMS的细胞成分也参与了L链。此两条迁移流的细胞成分包括GFAP阳性的成熟星状胶质细胞、Vimentin阳性的不成熟星形胶质细胞、Tuj-1阳性的成神经细胞，到达损伤部位可能起再生及修复作用。H、L链还表达神经营养因子bFGF和BDNF。结果提示bFGF和BDNF可能通过自分泌或旁分泌作用于其受体，不仅促进SVZ增殖同时也促进H链和L链细胞的迁移和分化，到达损伤区进行修复。3去大脑皮层血管对成年大鼠海马齿状回神经干细胞的影响本实验观察了去皮层血管对成年大鼠海马SGZ神经干细胞及bFGF表达的影响，结果显示去大脑皮层血管后成年大鼠SGZ中BrdU、Tuj-1、PCNA阳性细胞的数目增多、齿状回bFGF表达上调，相反Vimentin阳性细胞减少，结果提示与SVZ神经干细胞相比成年大鼠海马SGZ神经干细胞具有有限的自我更新和多向分化能力。4三七总皂甙对离体胚鼠皮层神经干细胞作用的实验研究选取E17胎鼠皮层细胞，通过原代细胞培养、传代、单细胞克隆和免疫细胞化学方法体外研究三七总皂甙对胚鼠皮层神经干细胞增殖、分化、自我更新及BDNF、bFGF表达的作用。结果显示，给药组原代、P1、P2细胞都能够形成较多的神经球；给药组P1细胞的单细胞克隆能够形成神经球；与对照组比较，给药组原代细胞培养4天Nestin、PCNA、<WP=4>Tuj-1、NF、Vimentin、GFAP、bFGF、BDNF阳性细胞均明显增多，阳性细胞能够形成明显的团状。结果提示体外三七总皂甙可能通过促进胚鼠皮层神经干细胞或前体细胞bFGF、BDNF自身合成增加（bFGF、BDNF通过自分泌或旁分泌）促进其存活、增殖、分化和自我更新。5三七总皂甙对去大脑皮层血管成年大鼠前脑BDNF、bFGF、Calbindin-D28K、PCNA表达作用的实验研究本研究观察了去皮层血管后成年大鼠前脑bFGF、BDNF、PCNA和Calbindin-D28K的表达以及三七总皂甙的作用，结果显示去皮层血管后bFGF、BDNF、Calbindin-D28K和PCNA在前脑包括皮层、海马、纹状体等脑区都有表达，损伤后期（15天和30天）bFGF主要表达在胶质细胞，BDNF和Calbindin-D28K表达在神经元。给予三七总皂甙后皮层、海马、纹状体阳性细胞的数量和反应强度都明显高于对模型大鼠，说明三七总皂甙促进去皮层血管后成年大鼠前脑神经元或胶质细胞表达bFGF和BDNF，bFGF和BDNF可能通过多种分泌方式及转运途径作用于受体促进损伤后神经元存活、损伤神经组织的修复，其机制包括上调Calbindin-D28K减轻神经元损伤、上调PCNA促进损伤DNA的修复。综上所述，本研究表明去皮层血管引起内源性bFGF、BDNF表达增多，bFGF、BDNF可能通过自分泌或旁分泌作用于受体，促进侧脑室SVZ、海马齿状回SGZ神经干细胞的增殖、分化，SVZ细胞还能形成迁移流到达皮层损伤部位，可能参与损伤的替代与修复。三七总皂甙对以上有明显的促进作用。三七总皂甙还能够促进去皮层血管后皮层、海马、纹状体中bFGF、BDNF在神经元和胶质细胞的表达，bFGF和BDNF可能通过多种分泌方式及转运途径作用于受体促进损伤后神经元存活、损伤神经组织的修复更多还原

【Abstract】 In recent years many research have proved that neural stem cells (NSCs) capable of self-renewal and multipotency persists in two adult mammalian brain regions: the subependymal zone (SVZ) of lateral ventricle and the subgranular zone (SGZ) in the hippocampal dentate gyrus. Neurotrophins such as basic fibroblast growth factor(bFGF) and brain-derived neurotrophic factor(BDNF) can promote the proliferation, migration and differentiation of NSCs, also have effects of on neuroprotection and neurorepair. We try to investigate the influences of cortical devasculating on the NSCs as well as the expressing of bFGF and BDNF in adult rat forebrain. Further we want to make sure the treatment effects of Panax Notoginseng saponins on it, and extend the foundation of modern medical science theories of the method of promoting tissue regeneration by removing blood stasis of Chinese medcine in treatment of brain disease.1 Influences of cortical devasculating on the NSCs in svz in adult rat and effects of Panax Notoginseng saponins on itOur research used adult Wistar rats with cortical devasculating on the left. BrdU was used ip to label the proliferating cells, and immunohistochemistry method was applicated to observe the influences of cortical devasculating on the proliferation, migration, and differentiation of NSCs and the expression of bFGF in SVZ,.In addition we observed the effects of Panax Notoginseng saponins on it. The results from serial sections showed that BrdU, PCNA, Nestin, Tuj-1 and Vimentin positive cells in SVZ from the caudal to the rostral of the lateral ventricle, including anterior horn, central part and inferior horn, were all increased. The reaction of them became stronger and the shape changed obviously after cortical devasculating. Tuj-1 mainly expressed in anterior horn of lateral ventricle, and glail cells were mainly located in the inferior horn of lateral ventricle. The expression of bFGF increased in SVZ at different level of the lateral ventricle, indicated that cortical devasculating induced the expression of it. bFGF might act on it’s receptor by autocrine or paracrine, so that stimulated the quiescent NSCs in SVZ to proliferate and differentiate. After treatment of Panax Notoginseng saponins, the expressions of all above increased and strengthen, indicated Panax Notoginseng saponins might promote the proliferation and differentiation of NSCs in SVZ through the mechanism of increasing the expression of endogenous bFGF after cortical devasculating. 2 Cortical devasculating induced the neural stem cells in subependymal zone to proliferate and form migrating stream to the lesion siteCortical devasculating not only accelerated the proliferation of NSCs in SVZ and RMS, but also induced the proliferation of SVZ in inferior horn of lateral ventricle in adult rat. The proliferating cells formed two chains (H chain and L chai) of migrating stream, and migrated to the cortical lesion site through corpus callosum.The H chain derived from the upperoir wall of inferior horn, and the L chain derived from the lateral wall of inferior horn. Cells coming from the RMS<WP=6>also participated in the L chain. Cell components of these two chains of migrating stream included GFAP positive mature astrocytes, Vimentin positive imature astrocytes, and Tuj-1 positive neuroblasts. These positive cells arrived the cortical lesion site and might contribute to regeneration and restoration. The H and the L chain also expressed bFGF and BDNF. These results indicated bFGF and BDNF might act on their receptors by autocrine or paracrine, and in turn not only accelerated the proliferation of SVZ, but also promoted the migration and differentiation of the H chain and the L chain. The H chain and the L chain arrived to lesion site participated in the repair. 3 Influences of cortical devasculating on the neural stem cells in hippocampal dentate gyrus in adult rat Our research observed the effects of cortical devasculating on the NSCs and the expression of bFGF in SGZ in adult rat. The results showed that the numbers of Br更多还原