不同方法共移植的嗅鞘细胞和雪旺氏细胞在损伤脊髓内的迁移和对轴突再生的影响

Migration and effect of cotransplantation of olfactory ensheathing cells(OECs)and Schwann cells(SCs)on axon regeneration in injured spinal cord

目的:观察经不同方法共移植的嗅鞘细胞和雪旺氏细胞在脊髓内的迁移特点和对轴突再生的影响。方法:将8只75±1d雌性SD大鼠随机分为2组,每组4只,利用NYU打击器制作T10脊髓损伤模型,打击高度25mm,打击杆重量10g。造模后2周,一组采用联合移植,雪旺氏细胞移植于损伤部位中心,嗅鞘细胞移植于距离损伤中心0.5mm处的头侧和尾侧的脊髓中线上;另一组采用混合移植,将嗅鞘细胞和雪旺氏细胞混合后移植于距离损伤中心0.5mm处的头侧和尾侧的脊髓中线上。每个部位注射4个点,深度为1.75mm、1.25mm、1mm、0.5mm,注射速度为0.1μl/min。联合移植组嗅鞘细胞量每点0.5μl含5×104个,雪旺氏细胞量为每点1μl含105个雪旺氏细胞;混合移植组为每点1μl含嗅鞘细胞和雪旺氏细胞各5×104个。细胞移植后1周和8周时各组分别取2只大鼠,以损伤部位为中心取包含细胞移植部位的脊髓,荧光和共聚焦显微镜下观察细胞迁移情况,利用神经丝(neurofilment,NF)和生长相关蛋白-43(growth associated protein-43,GAP-43)免疫荧光染色观察移植细胞对轴突再生的影响。结果:两组中均可见嗅鞘细胞迁移,主要在灰质和白质内沿脊髓纵轴向损伤部位迁移,还分别有一小部分沿中央管和蛛网膜下腔迁移;但雪旺氏细胞仅在与嗅鞘细胞混合移植于距离损伤中心0.5mm处的头侧和尾侧脊髓时才可见有限距离的迁移。联合移植时,NF阳性(NF+)和GAP-43阳性(GAP-43+)纤维伴随嗅鞘细胞迁移而沿脊髓纵轴延伸,雪旺氏细胞移植处可见NF+纤维从各个方向长入损伤部位(移植部位)并互相缠绕;混合移植时,大量NF+纤维伴随移植细胞迁移而延伸,损伤部位虽然NF+纤维较少,但没有互相缠绕现象。结论:雪旺氏细胞在损伤脊髓内迁移能力差;嗅鞘细胞不仅具有良好的迁移能力,而且可促进雪旺氏细胞迁移。无论是联合移植还是混合移植,移植细胞均能促进轴突再生,但联合移植时雪旺氏细胞移植处再生纤维互相缠绕、无法延伸。

Objective：To investigate varied migration and effect of cotransplantation of olfactory ensheathing cells（OECs）and Schwann cells（SCs）on axon regeneration in injured spinal cord of rats.Method：Spinal cord contusion at T10 segment was induced in adult female SD rats（n=8,75±1d）by NYU impactor at the height of 25mm,and all animals were assigned as follows：（1）GFP ＋OECs cotransplantation 0.5mm cephalad and caudally to the injured site combined with injection of SCs labelled by hochest at injured site （2）Mixed OECs and SCs（1：1）transplantation 0.5mm cephalad and caudally to injured site after 2 weeks（cell density：1×105/μl,depth：1.75mm,1.5mm,1.0mm,0.5mm,dosage：0.5μl cell suspension/per depth）.The rats were sacrificed at 1th and 8th week after transplantation respectively,and the T8-11 cord containing lesion and transplantation site were dissected,sectioned and stained immunochemically for fluorescent microscopy and then analsized for cell migration and axon regeneration.Result：After cotransplant...更多ation of OECs and SCs,SCs remained largely at the transplanted site,while OECs were capable of migrating from injection site to injured site longitudinally and laterally.Most of the OECs migrated to the injured site in gray matter and white matter,and some migrated through central canal or pia from the entry site.In transplantation of OECs and SCs to same site,OECs could migrate to the injury site,and SCs could migrate in a limited distance.After combined transplantation of OECs and SCs,NF＋ and GAP-43＋ axons were observed migrated parallelly to the OECs processes,transplantation with SCs contained a greater concentration of NF＋ axons,but no GAP-43＋ axons was evidenced.NF＋ axons penetrated into the SCs grafts from all directions and twisted irregularly in the injured site.After transplantation of OECs and SCs into same site,numerous NF＋ and GAP-43＋ axons parallel to mixed cells processes were observed with the density of NF＋ axons being low in injured site.Conclusion：Both mixed and combined transplantation of OECs and SCs has the capacity for cell migration and axon regeneration in contused spinal cord of rats,while the former is better than latter due to the good growth protocol.