神经干细胞移植延缓失神经肌肉萎缩的实验研究  

EXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE ATROPHY

作　　者：沈云东[1] 徐建光[1] 徐文东[1] 徐雷[1] 陆九州[1] 顾玉东[1] 

机构地区：[1]复旦大学附属华山医院手外科,上海200040

出　　处：《中国修复重建外科杂志》2008年第9期1051-1055,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30672124);上海市科学技术委员会基础重点资助项目(07JC14008)~~

摘　　要：目的研究异体神经干细胞(neural stem cell,NSC)移植于切断的周围神经远端,延缓失神经肌肉萎缩的作用,并探讨其发挥作用的可能机制。方法取2只孕12～14d绿色荧光蛋白(green fluorescent protein,GFP)转基因大鼠,取其胚胎并体外分离培养脊髓NSC。选取2月龄健康成年F344雌性大鼠32只,体重(180±20)g,随机分为实验组和对照组(n=16)。于右股部膝关节上1.5cm处水平切断胫神经及腓总神经,近端反折缝合,建立小腿三头肌失神经支配模型。实验组:将制备的5μLGFP-NSC悬液自胫神经远侧断端进针1cm后缓慢注入胫神经内;对照组:同法注入等量NSC培养上清液。术后观察大鼠一般情况,于术后4周及12周取材,测量小腿三头肌湿重,行肌肉HE染色、Mallory三色染色及突触后膜染色,观察并测量肌纤维横截面积维持率及肌肉突触后膜的形态和面积。结果各组大鼠伤口均Ⅰ期愈合,右侧后肢无溃疡发生。术后4周和12周,右侧小腿三头肌湿重,实验组分别为(0.849±0.064)g和(0.596±0.047)g,对照组分别为(0.651±0.040)g和(0.298±0.016)g,同时间点组间比较差异有统计学意义(P<0.05)。术后4周与12周,骨骼肌纤维横截面积维持率实验组分别为72.55%±8.12%和58.96%±6.07%,对照组分别为50.23%±4.76%和33.63%±4.41%;实验组均优于对照组,差异有统计学意义(P<0.05)。失神经肌肉经Mallory三色染色,术后4周可见肌纤维之间已有大量胶原纤维增生;术后12周,胶原纤维进一步增多,大部分肌肉纤维被取代,但实验组纤维化程度轻于对照组。术后12周,实验组突触后膜面积为(137.29±29.14)μm2,更接近于正常(198.63±23.11)μm2,达对照组(61.03±11.38)μm2的2倍以上,各组差异均有统计学意义(P<0.05)。结论异体胚胎脊髓NSC体内移植可发挥延缓失神经肌肉萎缩和维持失神经肌肉突触后膜形态、功能的作用,为临床上周围神经损伤后肌肉萎缩的防治提供了新的思路和方法�Objective To observe the delaying effect of neural stem cell （NSC） transplantation on denervated muscle atrophy after peripheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the spinal cords of green fluorescent protein （GFP） transgenic rats aged 12-14 days mechanically and were cultured and induced to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed （180 ± 20） g, were randomized into two groups （n=16 per group）. The animal models of denervated musculus triceps surae were established by transecting right tibial nerve and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 uL of GFP-NSC suspension and 5 uL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general condition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by first intension, no ulcer occurred in the right hind limbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps surae was （0.849 ± 0.064） g and （0.596 ± 0.047） g in the experimental group, respectively, and was （0.651 ± 0.040） g and （0.298 ± 0.016） g in the control group, respectively, showing a significant difference （P 〈 0.05）. The fiber section area of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ± 4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them （P 〈 0.05）. Mallory trichrome staining of muscle notified that there was more

关 键 词：周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植 

分 类 号：R651.3[医药卫生—外科学] Q813.1[医药卫生—临床医学]