【作者】 刘静；

【导师】 刘宏亮；

【作者基本信息】 第三军医大学 ， 康复医学与理疗学， 2009， 硕士

【摘要】 一、研究背景脊髓损伤(spinal cord injury , SCI)是指由于各种外力作用于脊柱所造成的脊髓压迫或断裂,多系车祸、坠落伤等造成脊柱脱位、骨折所致。脊髓是感觉、运动和植物神经系统的传导通路,因此损伤的主要症状是由于传导通路的中断而引起的麻痹,主要表现为损伤平面以下运动、感觉和自主神经功能的障碍。SCI所致瘫痪给患者本人、家庭和社会带来沉重的负担和严重的社会问题。随着科学研究的进展,神经干细胞移植从促进损伤脊髓再生与修复的目的出发,为SCI的治疗带来了新的希望。神经干细胞( neural stem cells, NSCs)是能主要分化为神经细胞的一种干细胞,广泛存在于神经系统,研究显示成年嗅球、皮层、室管膜层和室管膜下层、纹状体、海马齿状回颗粒细胞下层等脑组织中都分布着神经干细胞。神经干细胞终身具有自我更新能力和多向分化潜能,在一定条件下能分化为神经元、星形胶质细胞和少突胶质细胞。神经干细胞不仅能促进神经元的再生和脑组织的修复,而且通过基因修饰还可用于神经系统疾病的基因治疗,为许多难以治疗的神经系统疾病提供了新的治疗途径。神经干细胞在体内外的分化受到多种因素的影响,其最终分化结果与其所处的微环境是密切相关的。神经干细胞的移植为中枢神经系统疾病的治疗带来了新的希望,但移植治疗的关键问题是诱导神经干细胞定向分化所需的神经元,因此探求诱导神经干细胞定向分化的最佳微环境成为研究的热点。国内外研究表明,中药不仅可以保护神经细胞,提高神经元抵抗损伤的能力,而且能促进NSCs的增殖并诱导其定向分化为功能性神经元,达到治疗神经功能缺损疾病的目的。因此,研究中药对NSCs的增殖、分化及抗损伤的机制,对利用中药开发神经干细胞在神经系统疾病的应用具有重要意义。近年来中药单体在中枢神经系统功能损害性疾病的治疗方面显示了独特的疗效。有学者发现银杏叶提取物银杏内酯B(Ginkgolide B,GKB)对神经干细胞向神经元分化有促进作用。进一步研究发现,银杏内酯B还具有类似BDNF诱导神经干细胞分化为神经元的作用。黄芪皂甙( Saponins of Astragalus, SA)为黄芪的主要成分,研究表明黄芪注射液诱导体外培养的NSCs向神经元的分化有积极作用,但是单体黄芪皂甙对神经干细胞分化的作用未见相关报道。进一步阐明银杏内酯B及黄芪皂甙在神经干细胞分化的作用及机制,以及在神经干细胞修复脊髓损伤中的作用对神经修复领域具有重要价值。GFP转基因小鼠具有遗传稳定、全身细胞均表达绿色荧光蛋白,从其体内分离出的细胞不因细胞分裂降低绿色荧光表达强度,较好地解决细胞标记的问题,非常便于活细胞在生物体内生物学行为的观察,因而在细胞及基因治疗的研究中有较高的应用价值。二、目的通过体外培养昆明小鼠胎鼠神经干细胞,采用免疫荧光细胞化学方法比较银杏内酯B及黄芪皂甙对神经干细胞分化的影响;体内移植GFP转基因小鼠神经干细胞进入小鼠脊髓损伤部位,采用行为学和形态学方法观察移植到体内的神经干细胞在修复脊髓损伤中的作用。为探讨中药促进神经干细胞分化而治疗脊髓损伤的研究提供基础。三、方法1.从昆明小鼠胎鼠中分离和培养NSCs,在加入40mg/L银杏内酯B和20mg/L、40mg/L、60mg/L黄芪皂甙的分化培养基中培养24h、3d和7d,用倒置相差显微镜测量分化细胞神经突起长度;用免疫荧光细胞化学染色方法检测其微管相关蛋白(β-Tubulin)、胶质纤维酸性蛋白(GFAP)及少突胶质细胞特异性蛋白(CC-1)的表达,并计算阳性细胞的百分率;比较银杏内酯B和黄芪皂甙对神经干细胞的分化作用。2.将GFP转基因小鼠NSCs移植入小鼠脊髓全横断处,用形态学方法观察移植细胞在体内的分化情况,并用行为学方法检测脊髓功能的恢复情况,探讨NSCs移植在SCI恢复中所起的作用。在体实验分为三组:A.对照组:动物只施行切皮、打开椎管,不施行SCI;B.手术对照组:T11节段脊髓全横断损伤;C.NSCs移植组:在T11节段脊髓完全横断后立即用微量注射器将GFP神经干细胞注入横断部位。通过免疫组织化学观察移植神经干细胞在脊髓内的存活和分化,通过对各组小鼠进行后肢BBB运动功能评分观察小鼠脊髓功能恢复情况。四、结果1.从昆明小鼠胎鼠中成功分离和培养NSCs,Nestin染色阳性。2.银杏内酯B组分化(1d、3d)突起长度相比对照组突起长度有显著增长(P<0.05)。20mg/L、40mg/L、60mg/L黄芪皂甙组突起长度相比对照组突起长度均有显著增长(P<0.05)。银杏内酯B组突起长度比黄芪皂甙各组的突起长度均更长,具有显著性差异(P<0.05),20mg/L、40mg/L、60mg/L黄芪皂甙组神经突起的平均长度三组间比较无显著差异(P>0.05)。3.银杏内酯B组分化(3d、7d)后,β-Tubulin阳性神经元百分率、GFAP阳性星形胶质细胞百分率均显著高于对照组(P<0.01),CC-1阳性少突胶质细胞百分率无明显变化。20mg/L黄芪皂甙组分化为β-Tubulin阳性神经元百分率与对照组相比无显著性差异(P>0.05),40mg/L和60mg/L黄芪皂甙组分化为β-Tubulin神经元的百分率与对照组相比显著增高(P<0.01),40mg/L和60mg/L黄芪皂甙组分化为β-Tubulin神经元的百分率与20mg/L黄芪皂甙组相比显著增高(P<0.01),两种浓度组间无显著性差异(P>0.05)。20mg/L、40mg/L和60mg/L黄芪皂甙的三个浓度组分化为GFAP阳性星形胶质细胞百分率与对照组相比均无显著性差异(P>0.05),黄芪皂甙的三个浓度组间无显著性变化(P>0.05)。20mg/L、40mg/L和60mg/L黄芪皂甙的三个浓度组分化为少突胶质细胞百分率与对照组相比均无明显影响,黄芪皂甙的三个浓度组间无显著性变化(P>0.05)。4.在体研究发现,移植的NSCs不仅能够存活,而且NSCs移植治疗小鼠脊髓损伤后,能够分化为神经元,提示其对损伤脊髓的结构具有一定的修复作用。5.通过后肢BBB运动评分,观察到NSCs移植SCI小鼠脊髓后,对后肢整体运动功能修复具有明显的促进作用。五、结论1.银杏内酯B和黄芪皂甙均能促进突起的生长,但以银杏内酯B的作用更明显;银杏内酯B和黄芪皂甙均能促进NSCs向神经元分化,其中黄芪皂甙能促进NSCs向神经元定向分化,而银杏内酯B同时又促进了NSCs向星形胶质细胞的分化,两种中药对分化为CC-1阳性少突胶质细胞的百分率无显著影响。上述结果为进一步联合应用银杏内酯B(更好地促进突起生长作用)和黄芪皂甙(促进神经元定向分化)治疗脊髓损伤提供了可能性。2.通过体内移植GFP转基因小鼠神经干细胞进入小鼠脊髓损伤部位,NSCs移植到小鼠损伤脊髓后能分化为神经元并促进其功能的部分恢复。为下一步研究银杏内酯B及黄芪皂甙在NSCs移植治疗脊髓损伤的应用提供基础。更多还原

【Abstract】 Background:Spinal cord injury (SCI) is one of critical trauma in central nervous system (CNS), often caused by spinal column dearticulation or bone fracture after car accident and crash. Since the spinal cord is the transduction pathway of the movement, sensation and autonomic nervous system, the main symptom of SCI is the palsy due to the interuption of this transduction pathway. It principally represents as the disorder of movement, sensation and function of autonomic nerve under injury level. The palsy due to spinal cord injury brings heavy burden to the patient himself, his family and society, and also causes severe social problems sometimes. Fortunately, with the advancement of scientific research, the transplantation of neural stem cells (NSCs) which improves regeneration and reparation of spinal cord, gives a new hope for the treatment of SCI.NSCs is a sort of stem cells which could differentiate into nerve cells, they are extensively present in the obfactory bulb, the cerebral cortex, the ventricular zone subventricular zone, the corpora striata and the dentate gyrus of hippocampus. These cells have the potentiality of self-renew and multi-directional differentiation. NSCs can not only facilitate the regeneration of neurons and reparation of brain tissue, but also differentiate into neurons, astrcytes and oligodendrocytes in some conditions. The differentiation of NSCs in vivo and in vitro is affected by various factors, and the outcome of differentiation is closely related with microenvironment. Although the transplantation of NSCs brings a hope of the treatment of CNS diseases, the key point is how to induce NSCs to definitely differentiate into the neurons we want. In this way, the optimal microenvironment for orient differentiation has become hot in this field. Previous studies showed that the traditional Chinese medicine could not only prevent the nerve cells from injury, but also promote the proliferation of NSCs and induce them differentiate into functional neurons to cure the disease of the nerve impairment. Thus it is interesting to study the mechanism of the proliferation and differentiation of the NSCs, and the transplantation of NSCs may be a potential way to treat the disease of the CNS.Recently, traditional medicine monomers (the effective chemical components extracted from Chinese crude herbs) exert a distinct curative effect on the therapy of functional impairment of CNS. Evidence shows that ginkgolide B (GKB) facilitates NSCs to differentiate into neurons. Further researches discovered that GKB shares a similar ability as BDNF in promoting NSCs differentiate into neurons. It was reported that in vitro NSCs were induced to differentiate into neurons when Astragalus mongholicus injection was added in the culture medium. However, the effect of Saponins of Astragalus (SA), the essential component of Astragalus mongholicus on NSCs differentiation remains unclear. It is of great value to elucidate the effect and mechanism of GKB and SA in NSCs differentiation and repair of spinal cord injury. GFP transgenic mouse is a bred of mice with genetical stability to express green fluorescence protein in all cells. The cells isolated from the GFP transgenic mouse expressed the same fluorescence intensity as in vivo, although the cells divided for many times. So GFP transgenic mouse is an ideal model for the research on the biological features of living cells in vivo, and allow new approaches to life science research that general cell culture techniques cannot deliver.Objective:The effects of GKB and SA on NSCs differentiation was investigated in the NSCs derived from Kuming mice fetuses by immunofluorescence method. NSCs derived from GFP transgenic mouse was transplanted into the mice after spinal cord injury, the biological behaviors of the mice and the morphological changes were observed for the effects of the two traditional medicine monomers in this process.Methods1. NSCs, isolated from Kunming mice fetuses, were cultured in the medium pre-supplemented with 40 mg/L GKB or 20, 40 and 60 mg/L SA for 24 h, and 3 and 7 d. The length of the processes of these differentiated cells was observed under and inverted phase contrast microscope. The expressions ofβ-Tubulin, GFAP and CC-1 were detected by immunofluorescence staining, and the percentage of the positive cells was recorded. The effect of GKB and SA on the NSCs differentiation was compared.2. In vivo experiment, there were 3 groups, sham operation group, SCI group and NSCs transplantation group. The SCI animal model was established by opening vertebral canal and entirely cutting off T11 segment of spinal cord. In the NSCs transplantation group, the animals received a microinjection of NSCs derived from GFP transgenic into the lesion immediately after the injury. The growth, integration and differentiation of the transplanted NSCs were observed with immunohistochemical methods for the neural pathological changes. Hind limb BBB functional assessment was performed to evaluate the spinal cord function.Results1. NSCs were successfully isolated from Kunming mice fetuses, and cultured. These NSCs were identified to be Nestin positive.2. The length of processes in the GKB groups (1 and 3 d), were significant longer than that of control groups (P<0.05). The length of processes in the SA groups (20, 40, and 60 mg/L) were significant longer than that control groups (P<0.05). And the length in the three SA groups were significant shorter than that of GKB groups (P<0.05), but there is no significant difference among the three different-dose groups (P<0.05).3. The percentages ofβ-Tubulin+ neuron-like cells and GFAP+ astrocytes in GKB groups were significant larger than control groups (P<0.01), and the percentage of CC-1+ oligodendrocyte-like cells had no marked change compared with control groups. The percentages ofβ-Tubulin+ neurone-like cells in 20 mg/L SA group had no significant difference with control groups, and the percentages of these cells in 40 and 60 mg/L SA groups were significant larger than control groups (P<0.01), and 40 and 60 mg/L SA groups had notable increase in comparison with 20 mg/L SA group (P<0.01), but there was no difference between 40mg/L and 60mg/L SA groups (P>0.05). The percentages of GFAP+ astrocytes in three SA groups had no significant difference with control groups (P>0.05). So did the percentages of oligodendrocytes in SA groups. There was no marked change among these three SA groups (P>0.05).4.In vivo study showed the NSCs which transplanted into the impaired spinal cord could survive more than one month and differentiated into neurons, this result suggested that the transplanted NSCs could at least partly repair the the impaired spinal cord.5 . The data of the hind limb BBB functional assessment indicated that the transplantation of NSCs obviously improved the motor function of the injured hind limb. Conclusion1. Both GKB and SA have the effect to improve the growth of nerve processes in isolated NSCs, but the effect of GKB is more significant. Both of them can induce the NSCs to differentiate into neurons, but GKB also improves the differentiation into astrocytes. There was no significant difference in the CC-1+ oligodendrocyte-orientation differentiation between the two traditional medicine monomers. Our results suggest that combined using these two monomers will be beneficial for the treatment of spinal cord injury. GKB is better for improving the growth of nerve processes, and SA is only to induce the differentiation of NSCs.2. In vivo study indicates that the transplantation of GFP transgenic mice NSCs into impaired spinal cord could promote the reparation of the function of spinal cord. Our research suggests that GKB and SA are able to promote the spinal cord injury reparation, but further study is needed.更多还原