脂肪干细胞及其来源细胞外基质构建的新型组织工程神经在周围神经修复中的应用

发布时间：2018-05-23 23:15

本文选题：脂肪干细胞 + 细胞源性细胞外基质　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：对于临床中大段神经缺损,目前商用的神经移植物难以获得满意的修复效果。大量的研究证实细胞外基质对调控细胞行为具有重要意义,无论是对干细胞还是神经再生相关细胞,均可以促进其迁移、增殖和分化。所以,对于周围神经而言,细胞外基质微环境可能对周围神经的再生具有良好的促进作用。脂肪干细胞可通过临床抽脂等获取,其来源相对广泛,被广泛用于组织工程与神经再生研究,但研究的关注点多放在它的分化能力、旁分泌作用上,而对其分泌的细胞源性细胞外基质研究不多。目的：本课题通过研究脂肪干细胞促进神经再生的作用,进一步探讨脂肪干细胞来源细胞外基质微环境对神经再生的影响,并利用脂肪干细胞来源细胞外基质修饰生物材料支架并复合脂肪干细胞构建一种新型组织工程神经并评价其修复周围神经缺损的效果。方法：1.通过酶消化的方法培养原代脂肪干细胞(adipose-derived stem cells, ADSCs),然后进行流式表面抗原的鉴定、三系诱导分化、CCK8法测定不同代ADSCs的增殖及向类雪旺细胞分化能力;2.ELISA法测定ADSCs条件培养基中神经再生相关因子的含量。EdU法测定雪旺细胞在加入条件培养基和与ADSCs间接共培养后的增殖情况。免疫荧光测定背根神经节神经元在加入条件培养基、与ADSCs间接共培养和直接共培养的轴突生长长度；3.制备ADSCs源性细胞外基质,用光镜和电镜鉴定其表面特征,免疫荧光鉴定其成分,体外评价其对雪旺细胞和背根神经节的作用；4.利用ADSCs源性细胞外基质修饰壳聚糖空管内表面并复合ADSCs构建组织工程神经,并将之植入到大鼠坐骨神经缺损模型中,并从步态分析、电生理评价、组织学评价和靶器官评价等方面评价其促进周围神经修复的作用。5.对比血管基质细胞混合物(stromal vascular fraction. SVF)与ADSCs在大鼠坐骨神经缺损修复中的作用。结果：1.脂肪干细胞诱导的类雪旺细胞,S100、GFAP和p75免疫荧光染色阳性；2.在与ADSCs间接共培养环境下,间接共培养组的EdU阳性细胞与条件培养基一致,高于普通培养基,差异有统计学意义(P0.01)；在与ADSCs间接共培养和直接共培养的环境下,直接共培养组的背根神经元轴突长度常于其它组,差异有统计学意义(P0.01)；3.制备了ADSCs源性的细胞外基质,其表面的基质成分交织成网,高倍电镜下可见大量微孔样结构。其成分含有大量的层粘连蛋白、纤连蛋白、Ⅰ和Ⅳ型胶原。体外验证了ADSCs源性细胞外基质同样具有促进雪旺细胞增殖和背根神经节轴突生长的作用；4.构建的ADSCs源性细胞外基质修饰并复合ADSCs勺组织工程神经在体内促进了模型大鼠步态、复合肌肉动作电位、有髓神经纤维直径和髓鞘厚度及靶肌肉横截面肌纤维面积等指标的恢复,结果优于ECM和ADSCs组,并接近自体神经的治疗效果；5.一与ADSCs相比,SVF也可使神经纤维直径和髓鞘厚度增加,并提高腓肠肌湿重恢复率,两者之间差异没有统计学意义,但是高于空管组而低于自体神经,差异有统计学意义(P0.05)。结论：1. ADSCs可以向类雪旺细胞分化;2.ADSCs的旁分泌作用可促进雪旺细胞的增殖和背根神经元轴突的生长,在神经元轴突生长的过程中,细胞与神经元轴突的接触也发挥了重要的作用；3.ADSCs源性ECM在体外可促进雪旺细胞的增殖和背根神经节神经元轴突的生长,并可以引导雪旺细胞和轴突顺ECM的方向生长；4.构建的ADSCs源性细胞外基质并复合ADSCs的组织工程神经促进了SD大鼠坐骨神经功能和形态的恢复,其结果接近自体神经；5.SVF具有与ADSCs相似的促进SD大鼠坐骨神经再生的作用,可以作为替代ADSCs的种子细胞使用。
[Abstract]:Objective : To study the effects of extracellular matrix on the regeneration of peripheral nerve .
3 . preparing ADSCs source extracellular matrix , using light microscope and electron microscope to identify its surface features , and immunofluorescence to identify its components , and evaluate its effect on Schwann cells and dorsal root ganglia in vitro ;
4 . Using ADSCs to modify the inner surface of chitosan hollow tube and compound ADSCs to construct the tissue engineering nerve , and implant it into the rat sciatic nerve defect model , and evaluate its role in promoting peripheral nerve repair from gait analysis , electrophysiological evaluation , histological evaluation and target organ evaluation . 5 . Contrast the vascular matrix cell mixture ( stromal vascular fraction ) . SVF and ADSCs play a role in the repair of sciatic nerve defects in rats . Results : 1 . Fat stem cell - induced Schwann cells , S100 , GFAP and p75 immunofluorescence staining were positive ;
2 . In the indirect co - culture environment with ADSCs , the EdU positive cells of the indirect co - culture group were consistent with the conditioned medium , which was higher than that of common medium ( P0.01 ) .
In the environment of indirect co - culture and direct co - culture with ADSCs , the axon length of dorsal root neurons of the direct co - culture group was higher than that in other groups ( P0.01 ) .
3 . The extracellular matrix of ADSCs derived from ADSCs was prepared . The matrix composition on the surface of ADSCs was made into a net , and a large number of micro - pore - like structures were observed under electron microscope . The components contained a large amount of laminin , fibronectin , I and IV collagen .
4 . The reconstructed ADSCs derived extracellular matrix modified and compound ADSCs spoon tissue engineering nerves promoted the restoration of gait , compound muscle action potential , myelinated nerve fiber diameter , myelinated nerve fiber diameter , myelinated nerve fiber diameter and muscle fiber area of target muscle in vivo . The results were superior to that of ECM and ADSCs and close to the treatment effect of autologous nerve ;
5 . Compared with ADSCs , SVF could increase the diameter of nerve fibers and the thickness of myelin sheath , and increase the recovery rate of wet - weight and wet weight , but there was no significant difference between them . However , the difference was statistically significant ( P0.05 ) . Conclusion : 1 . ADSCs can differentiate into Schwann cells ; 2 . The side - secretion of ADSCs can promote the proliferation of Schwann cells and the growth of dorsal root neurons . During the axon growth of neurons , the contact between cells and axons also plays an important role .
3 . The proliferation of Schwann cells and axonal growth of dorsal root ganglion neurons can be promoted in vitro by ADSCs .
4 . The tissue engineering nerve of ADSCs derived from ADSCs promotes the recovery of sciatic nerve function and morphology in SD rats .
5.SVF has the function of promoting the regeneration of sciatic nerve of SD rats similar to that of ADSCs , and can be used as seed cells for the replacement of ADSCs .
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R651.3

【相似文献】