嗅鞘细胞移植到大鼠损伤脊髓前后的形态学研究

Morphological study of OECs before and after transplantation into injured spinal cord of rat

目的:观察体外培养和植入损伤脊髓的自发绿色荧光嗅鞘细胞(Green fluorescent protein-olfactory ensheathing cells,GFP-OECs)的形态结构特征,研究绿色荧光蛋白(Green fluorescent protein,GFP)及脊髓损伤的病理环境对其形态的影响。方法:用酶消化法分离培养来自2.5月龄的绿色荧光蛋白转基因大鼠嗅球最外层的嗅鞘细胞,对培养细胞定期进行光镜、电镜及激光共聚焦显微镜观察并摄片,观察其在体外培养的形态特征。培养第10 d,用显微外科技术将嗅鞘细胞移植到脊髓挫伤后的T10节段,分别在移植术后7、15 d取材,观察细胞植入损伤脊髓后光电镜下形态。结果:GFP-OECs在荧光显微镜下呈绿色强荧光,培养第7 d,为双极样(梭形或纺锤形)、三极样和扁圆形3种典型的细胞形态,以双极梭形为主;培养第10 d,细胞形态为梭形,植入损伤脊髓后,移植细胞胞体呈长梭形,一端突起细长,与另一嗅鞘细胞接触,呈条索状生长。电镜下嗅鞘细胞核不规则,胞质内有丰富的粗面内质网、游离核糖体及线粒体。结论:GFP不影响嗅鞘细胞(Olfactory ensheathing cells,OECs)形态。在脊髓损伤处,OECs胞体呈长梭形,突起细长,与培养10 d移植时比较,形态无显著改变,早期脊髓损伤病理环境不影响GFP-OECs形态。

Objective：To observe the morphological features ofgreen fluorescent protein-olfactory ensheathing cells（GFP-OECs）cultured in vitro and transplanted into injured spinal cord of rats,and study the influence of pathological environment of spinal cord injury and GFP on their morphology. Methods：Outermost olfactory ensheathing cells were isolated from the olfactory bulbs of adult green fluorescent protein transgenic rats,OECs were cultured by enzyme digestion.The cells were observed and photographed regularly with light microscope,laser scanning confocal microscope and transmission electron microscope before transplantation in order to observe their morphological characteristics in vitro culture. When the cells were cultured in medium for 10 days,the cells were transplanted into contusion area of T 10 spinal cord in SD rat with microsurgical technique. The GFP-OECs in spinal cord injury were observed with light and electron microscopes after 7 and 15 days. Results：After primary cultured for 7 days,strong green fluorescence staining of GFP-OECs were observed,GFP-OECs appeared three typical morphology：dipolar,tripolar and oblate,but mainly dipolar.After primary cultured for 10 days,major cells were dipolar. After transplanted into injured spinal cord,the cells were dipolar,the nuclei were irregular,and there were rich rough endoplasmic reticulums,free ribosomes and mitochondrion in the cytoplasm. Conclusion：The morphology of OECs is not effected by GFP. There is no significant morphological difference of GFP-OECs before and after transplantation,the appearance of GFP-OECs do not change in injured spinal cord in its early stage.