Myroilysin脱细胞神经支架对牙髓干细胞增殖体外神经分化的影响

发布时间：2018-02-01 12:20

  本文关键词： Myroilysin 神经支架 牙髓干细胞 神经分化　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:观察深海细菌酶Myroilysin水解法制备的脱细胞神经支架对牙髓干细胞增殖及体外神经分化的影响。材料和方法:1.使用酶消法从年轻第三磨牙牙髓组织中提取人牙髓干细胞,经有限稀释法纯化扩增培养,作为本实验中与无细胞神经支架结合的种子细胞。2.分别运用Myroilysin水解法和传统的Sondell化学法脱除大鼠坐骨神经内的原生细胞制备无细胞支架。通过cck-8法测定两种支架及其制备过程中的残留成分对种子细胞增殖的影响。3.分别用免疫荧光染色和免疫印迹杂交实验western blotting检测两种支架对种子细胞向神经方向分化的影响。结果:1.经分离、纯化和扩增培养,所获得的人牙髓干细胞具有很强的增殖分裂活性,间充质干细胞标志性蛋白STRO-1呈阳性,神经微管蛋白β-Ⅲtubulin经免疫荧光染色法检测呈阳性。2.较高浓度的Myroilysin或脱氧胆酸钠(作为Sondell化学法的主要洗脱剂)都有使牙髓干细胞密度显著降低的不利作用;而两者在低浓度情况下,Myroilysin对种子细胞增殖活性的抑制明显低于脱氧胆酸钠。分别使用两种不同支架的浸润提取液培养牙髓干细胞时,早期Myroilysin酶支架组的细胞增殖活性高于Sondell化学支架组,后期两者都可达到较高的细胞密度。3.牙髓干细胞体外神经诱导分为四组:单纯神经诱导液组、神经诱导液+化学法神经支架组、神经诱导液+酶法神经支架组,无神经诱导液的普通培养基组作为阴性对照。免疫荧光染色显示:三个诱导组的细胞干细胞标志性蛋白STRO-1染色从有到无,微管蛋白β-III tubulin和神经胶质细胞标志蛋白GFAP在诱导前后都有表达,神经元标志蛋白NeuN表达量明显增高。免疫印迹杂交实验显示:GFAP在两个支架组表达增高,NeuN只在Myroilysin支架加神经诱导液组表达明显增高。结论:深海细菌酶Myroilysin的酶活性具有自限性和时效性,在酶水天然神经解制备脱细胞支架的过程中,酶效价呈时间依赖性降解。因此Myroilysin水解法制备的神经支架对种子细胞增殖活性的抑制作用很快消失,表现出良好的生物相容性。Myroilysin水解法制备的无细胞支架结构更为膨胀疏松,在更彻底的去除组织免疫原性的同时,保留了更多的原生结构,对种子细胞的漂移、附着、增殖、分化产生更好的地形诱导促进作用。
[Abstract]:Objective: to observe the effects of acellular neural scaffolds prepared by deep-sea bacterial enzyme Myroilysin hydrolysis on the proliferation and neural differentiation of dental pulp stem cells in vitro. 1. Human dental pulp stem cells were extracted from pulp tissue of young third molar by enzyme digestion. The culture was purified and amplified by limited dilution method. As seed cells combined with acellular nerve scaffolds in this experiment, the Myroilysin hydrolysis method and the traditional Sondell chemical method were used to remove the progenitor cells from the sciatic nerve of rats, respectively. Cell free stents. Effects of residual components of two scaffolds and their preparation on seed cell proliferation were determined by cck-8 method. Wester was detected by immunofluorescence staining and Western blot hybridization, respectively. N. Blotting was used to detect the effect of two scaffolds on the neural differentiation of seed cells. Purified and cultured, the obtained human dental pulp stem cells have strong proliferative and mitotic activity, mesenchymal stem cells iconic protein STRO-1 is positive. Tubulin 尾-鈪，

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