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) s7 y4 H: b0 Q; ? % O" f4 {2 ~# R+ g 【摘要】 [目的]建立大鼠脊髓全横断损伤模型,应用显微操作方法和人脐血干细胞移植修复大鼠脊髓损伤,观察显微操作技术对大鼠受损脊髓功能恢复的影响。[方法] 取足月健康顺产新生儿脐血,分离、培养脐血干细胞。60只SD雌性大鼠,随机分为假手术对照组(A组,n=10)、实验对照组(B组,n=10)、常规治疗组(C组,n=20)和显微治疗组(D组,n=20)。A组大鼠只打开椎板;B、C组大鼠采用常规方法行T8~9平面脊髓全横断,造成急性脊髓损伤,C组大鼠于脊髓两断端用1 μl注射器分别注射脐血干细胞悬液1 μl(干细胞浓度为6×109/L ~7×109/L),B组同法注射等量PBS液;D组采用显微操作技术行脊髓横断,并以口径50 μm的毛细玻璃针用相同方法注射脐血干细胞。术后1~8周内每周进行1次后肢BBB评分,术后第8周处死大鼠,对比观察术中和术后第8周各组大鼠脊髓大体标本外形、颜色、质地和体积大小改变。[结果]A组大鼠手术前后运动功能、脊髓大体标本未见明显变化。B、C、D组大鼠术后双侧后肢完全性瘫痪,B组大鼠无明显恢复。而C、D组大鼠,从术后第2周开始逐渐恢复部分后肢运动功能。第3周以后,D组大鼠后肢运动功能BBB评分明显优于C组,具有显著性差异(P+ C& |! t: n% Q
【关键词】显微操作; 脊髓损伤; 脐血干细胞; 移植1 w' C+ \7 o4 Q; y+ |: v1 n) y% T
Micromanipulation used in surgical procedure of transected spinal cord treated with umbilical cord blood stem cells grafts in rats∥YIN Wenhua,JIN Dadi,LU Kaiwu,et al.Department of Spinal Surgery,Southern Hospital Affiliated to Nangfang Medical University,Guangzhou 510515,China/ B7 \; Q# A, V+ @8 @0 ]5 }
7 C6 {6 O- R$ x4 H1 ?7 hAbstract:[Objective]To establish effective and reliable complete spinal cord transection models in rats, and to investigate the effects of micromanipulation used in surgical procedure of transected spinal cord treated with human umbilical cord blood stem cells (UCBSCs) grafts in rats.[Method]Human UCBSCs were obtained from umbilical cord blood of term deliveries.Sixty SpragueDawley (SD) female rats were divided randomly intogroup A(control group), group B(spinal cord transection and PBS injection), group C(spinal cord transection and UCBSCs transplantation) andgroup D(spinal cord transection and UCBSCs transplantation by using micromanipulation).All groups were operated, and 1 μl UCBSCs(6109/L ~7109/L) were injected into the both ends of the completely transected spinal cord at thegroups C and D by two different surgical procedures respectively. From 1 week to 8 week postoperation, a behavioral testing was performed weekly upon each hindlimb for all animals according to the BBB scoring system. At the 8th week, all animals were sacrificed and the spinal cords were taken out for morphological observation. [Result]No changes were observed at group A pre and postoperation. After 2 weeks postoperation, the hindlimb motor function of groups C and D began to recover. After 3 weeks postoperation, thegroup D showed more improvement thangroup C, and the difference betweengroups D and C were significant in the BBB scores(P: \5 N8 d8 t, @' c
) Q" { k; E$ o0 q/ l! H6 B研究表明[7],将人脐血干细胞植入大鼠脑内,移植细胞能够分化为神经胶质细胞和神经元。Willing等[8]将人脐血干细胞移植入成年大鼠局灶性脑缺血灶内24 h后,观察到成年大鼠记忆功能的改善。在本实验中,与采用人脐血干细胞移植治疗的C、D两组相比,虽然B组术中所见与脐血干细胞移植治疗的C组相似,但是,术后B组大鼠双侧后肢运动功能始终未见恢复,而且术后第8周,B组大鼠脊髓标本横断处两侧直径差异显著,远侧段脊髓明显萎缩、干瘪,直径变细,外形轮廓不规则。这表明,脐血干细胞移植对脊髓损伤有较好的治疗作用,但是其具体作用机制目前尚不完全清楚,有待进一步深入研究。: E6 j& z+ W$ W7 l
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图1a普通操作致脊髓背静脉和硬脊膜毛细血管网明显充血、扩张图1b采用显微操作的D组,脊髓背静脉清晰可见,硬脊膜毛细血管无充血图2aB组大鼠脊髓横断 PBS残端注射后,横断处被水肿、膨出的残端脊髓组织充填图2bC组行脊髓横断 脐血干细胞移植后,横断处两侧部分脊髓塌陷图2c、2dD组采用显微操作行脊髓横断 脐血干细胞移植后,硬脊膜保存完好,脊髓残端整齐、结构清楚,横断间隙均匀图3术后第8周脊髓大体形态学比较+ \4 U2 _3 K9 c
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图3aB组大鼠脊髓横断处毁损严重,局部可见长段瘢痕组织增生,远侧段脊髓明显萎缩外形干瘪,直径变细图3bC组大鼠脊髓横断处亦可见长段瘢痕增生,远侧段脊髓直径稍变细,但外形饱满、圆润图3cD组大鼠脊髓横断处局限性瘢痕组织增生,横断处两侧脊髓直径,外形无明显差别 ; H3 H9 m+ k1 j/ a+ o" H, p; j1 u- K7 O" {( L9 d# k" }; D7 M4 C
综上所述,本实验成功建立了脊髓完全性横断大鼠模型,在脐血干细胞移植治疗脊髓损伤的同时,采用显微操作技术明显提高了受损脊髓的神经功能恢复。随着对脐血干细胞移植修复机制的深入研究和移植治疗方法的不断改进,相信显微操作技术必将在神经损伤性疾病的治疗中发挥更大的作用。$ K% |- f- W% C& Y- F
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