【作者】 黄保胜；

【导师】 李立新；

【作者基本信息】 南京医科大学 ， 神经外科学， 2008， 硕士

【摘要】 目的:本研究旨在探索利用诱导分化的骨髓间充质干细胞（BMSCs）作为种子细胞,结合不同生物支架体外构建组织工程神经复合物移植修复大鼠脊髓损伤、评价移植治疗的效果并探讨其修复机制。方法:1,选取SD大鼠,体重约80g,无菌条件下取股骨骨髓,采用密度梯度离心与贴壁培养相结合的方法分离、纯化得到BMSCs,流式细胞仪检测BMSCs表面标志物。加入脑源性神经营养因子（BDNF）和全反式维甲酸（AT-RA）两种诱导分化剂,体外诱导BMSCs神经元方向定向分化7d和14d后,利用细胞免疫组织化学和神经电生理的方法鉴定分化的神经细胞。2,将BMSCs（第三代,P3）作为种子细胞,种植在多聚赖氨酸包被的聚乳酸-羟基乙酸（PLGA）和聚β羟基丁酸酯（PHB）两种生物支架上培养5d后,再加入BDNF和AT-RA神经方向诱导分化7d,体外分别构建出A和B两种组织工程神经复合物。用倒置相差显微镜观察细胞在支架上的生长和分化行为,应用免疫组织化学鉴定支架上培养的细胞。3,取成年SD大鼠28只,制作脊髓(T₁₀)右半切4mm长块状缺损模型,随机分成4组:实验A组损伤区移植组织工程神经复合物A（PLGA支架）;实验B组损伤区移植组织工程神经复合物B（PHB支架）:对照C组移植诱导分化的BMSCs;对照D组损伤未移植。移植后各组大鼠继续单笼喂养12周,每周均行BBB脊髓运动功能评分。伤后第12周予辣根过氧化物酶（HRP）神经逆行示踪评价脊髓感觉和传导功能的恢复情况,并取损伤处脊髓组织,显微镜下观察各组大鼠移植区的形态结构修复情况。对损伤区脊髓组织行免疫组织化学染色,分别比较各组NF,GAP-43,BCL-2以及MBP等蛋白的阳性表达情况探索损伤区脊髓功能修复的机制。结果:1,分离培养的贴壁细胞（P3）具有典型的成纤维细胞样形态和BMSCs细胞表面标志。BDNF联合AT-RA诱导后的细胞表达神经元特异性标志物神经元特异性烯醇化酶（NSE）和微管相关蛋白（MAP-2）,电生理功能检测可以发现反映神经细胞特性的动作电位和彼此形成突触联系的特征电流。2,倒置相差显微镜和扫描电镜观察结果显示:诱导分化后,发现部分细胞长出的神经轴突跨越支架孔隙的三维空间结构,布满支架的表面和孔隙,并彼此建立了突触联系。免疫组织化学鉴定为微管相关蛋白2（MAP-2）抗体阳性,表明体外构建的组织工程神经复合物中包含早期功能神经元,为该组织工程神经复合物移植修复脊髓损伤创造了条件。3,BBB评分结果显示:伤后2～12周,实验A组和B组大鼠BBB运动功能评分均较对照C组和对照D组明显提高（P＜0.05）,且实验A组高于实验B组（P＜0.05）,差异有统计学意义。伤后12周,显微镜下脊髓移植修复区形态结构观察:实验A组和实验B组损伤处脊髓组织完整性明显好于对照C组和对照D组,留下空洞较两对照组少,而实验A组的空洞又较实验B组少,形态结构修复好于实验B组,对照D组脊髓完整性中断,留下缺损最多。4,HRP神经逆行示踪显示:两实验组脑组织中均可见到较多的逆行HRP标记阳性神经元,且实验A组多于实验B组,而对照C组仅见少量HRP阳性神经元,对照D组没有见到HRP阳性神经元。5,免疫组织化学染色显示:两实验组移植区NF阳性神经元和GAP-43阳性轴索数量较多,而对照C组少,对照D组没有发现阳性神经元和轴索;标记神经元抗凋亡的BCL-2染色两实验组均有较多阳性染色,对照C组很少阳性染色,对照D组则无阳性染色;两实验组标记髓鞘形成的MBP蛋白表达较明显,且实验A组的MBP明显多于优于实验B组,而对照C组MBP蛋白表达不明显,对照D组未见MBP表达。结论:1,BDNF联合使用AT-RA是有效的BMSCs向神经元定向分化的诱导方法,所诱导得到的细胞具有神经元的形态和功能特征。2,生物支架支持骨髓间充质干细胞的生长和定向分化,支架复合种植诱导分化的BMSCs在体外构建组织工程神经复合物,为移植修复脊髓损伤创造了条件。3,体外构建的组织工程神经复合物移植体内后,促进了脊髓损伤区神经再生,形态结构的修复和运动功能的恢复。疗效明显优于单纯的BMSCs细胞移植;组织工程神经复合物的生物支架的选择影响对脊髓损伤的疗效,选用PLGA支架的复合物疗效优于选用PHB支架的复合物。更多还原

【Abstract】 Objective: To fabricate tissue-engineering neural complex with bone marrow mesenchymal stem cells(BMSCs) binding different kinds of biological scaffolds; to observe the effect of spinal cord injury on rats treated with transplantation of tissue-engineering neural complex, and to explore its mechanism. Methods: 1, BMSCs were isolated from bone marrow of rats with density gradient centrifugation and adherent culture. Expression of the BMSCs surface marker was detected by flow cytometer. BMSCs of P3 were induced and differentiated into neuron by applying brain-derived neurotrophic factor (BDNF) and alltransretinoic acid(AT-RA) for 7d and 14d. The appearance and function of the BMSCs after differentiation were characterized by Immunohistochemistry and neurophysiology. 2, BMSCs, which were used as seed cells, were inoculated onto the two kinds of biological scaffolds-polymer of lactic and glycolic acids(PLGA) and poly-3-hydroxybutyrate(PHB) coated with the polylysine. And then BDNF and AT-RA were added into the neural complex for 7d. Tissue- tissueing neural complex A and B were fabricated in vitro with PLGA and PHB respectively. The growth and differentiation of BMSCs implanted on biological scaffolds were observed by phase contrast microscope and Immunohistochemistry 3. Twenty eight SD rats were half-transected at T10 cord level and a 4mm segment caudal to the transaction was removed, and then assigned randomly into four groups: experiment group A(tissue-engineering neural complex A transplanted into the spinal cord gap), experiment group B(tissue-engineering neural complex B transplanted into the spinal cord gap), control group C (BMSCs injected into the spinal cord gap), and control group D(nothing positioned into the spinal cord gap). The rats were seeded in a single cage respectively for 12 weeks. The functional recovery and morphological repairing of the injured spinal cords were evaluated by the BBB scale、HRP tracing technique and Immunohistochemistry. Results: 1, The cultured BMSCs in P3 possess typical fibroblast-like morphology and cell surface antigen of BMSCs. The cells induced by BDNF and AT-RA exhibit neuronal phenotype, and show neuron specificenolase (NSE) and microtubule-associated protein-2(MAP-2) positive expression. And the characteristics of neurons induced by BDNF and AT-RA express the electrophysiology function. 2. Inverted phase contrast microscope and scanning electron microscope(SEM) were applying to observe the mophological changing in BMSCs: after neural differentiation of BMSCs induced by BDNF and AT-RA, formation of the three-dimensional network poly was covered with neurite contacting each other. Positive expressing of MAP-2 in BMSCs after differentiation was identified by immunohistochemistry. The tissue-engineering neural complexs were prepared qualificatedly for transplantation treatment of spinal cord injury. 3. The average BBB scales in two experiment groups were better than the control groups for all time points from 14 days(P < 0.05). Further more, BBB scales of the experiment group A was higher than the experiment group B(P <0.05). and the scales of the control group D was the lowest. After 12 weeks, Transplanted distinct in the injured spinal cord was observed by microscope. And the intaction of spinal cord in the experiment group A and the experiment group B was better than the two control groups, and the morphological repairing of the injured spinal cord in the experiment group A was better than that in the experiment group B. The spinal cord on the control group D was wholely discontinued. 4, More labeled motoneurons in contral brain of the experiment groups A and B were observed by utilizing HRP tracting technique, fewer in the control group C and no labeled motoneurons in the control group D. 5, More NF-positive neurons、GAP-43-positive axons and MBP-positive myelins were observed in the experiment groups A and B, fewer in control group C, and no positive neurons ,axons and myelins in the control group D. BCL-2 expression in the two experiment groups were higher than the two control groups, fewer in the control group C and no BCL-2 expression in the control group D. Conclusion: 1.The combination of BDNF and AT-RA is an effective method of neural differentiation for BMSCs. The cells induced by BDNF and AT-RA possess the appearance and function of neurons.2.The biological scaffolds can support the growth and differentiation of BMSCs. The tissue engineering neural complex can be fabricated in vitro by the biological scaffolds seeded with BMSCs.3. Transplantation of tissue engineering neural complex can promote neural and myelin regeneration, structural repairing and functional recovery of half-cut lump spinal cord injury, its curative effects are better than BMSCs transplatation alone. In addition, different scaffold materials have different curative effect on spinal cord injury in rats. The curative effective effect using PLGA for biological scaffold is better than that using PHB.更多还原