神经干细胞移植延缓失神经肌肉萎缩的实验研究

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沈云东 徐建光 徐文东 徐雷 陆九州 顾玉东, y. d" L$ X$ c( G4 o
【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
# ^6 c' y5 L8 N* _缩的作用，并探讨其发挥作用的可能机制。 方法 取2 只孕12 ～ 14 d 绿色荧光蛋白（green fluorescent protein，GFP）5 q' J2 k/ C# G; x9 ~5 i
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
% S7 k  v9 E% X1 _! w为实验组和对照组（n=16）。于右股部膝关节上1.5 cm 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
% |/ r6 F! y- f, s9 K9 u* K神经支配模型。实验组：将制备的5 μL GFP-NSC 悬液自胫神经远侧断端进针1 cm 后缓慢注入胫神经内；对照组：同法/ T/ I1 q8 v0 g- s
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、
+ q; r% M1 }5 O& O& XMal lory 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! t# r' Q1 }* A- g# B) O$ n! M( S8 G( R
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 F! }" J4 q+ d1 u
（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）。2 e' |' l) E9 o9 D
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% ±
! c1 T. P3 V) Q# ~" j% W4.76% 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* `2 p3 I( L/ e+ |( X: ^) s
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
! w+ y& K0 v' C4 g6 I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
7 j5 y. h0 P, H* d) T- D% {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4 H# M* ^2 C( g+ t
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" j3 ]7 R2 b$ Y, }- E& c6 G" z
的思路和方 法。
8 F" [5 L: [# s( ]& f0 P6 N  o【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植3 `- T6 J( f+ P0 U
中图分类号： R651.3 Q813.1 文献标志码：A; X8 ^; {) S# R7 C2 Q
EXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE4 H% B9 p# Y5 F
ATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,8 g6 c9 M& P9 {- \+ t: A$ z
Huashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@, K/ W$ v1 \$ @
hotmail.com7 Y. u% O+ z0 G& [7 ~! N
【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated
+ q6 i0 F  T" \( P* n3 U# Vmuscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the
. L, i7 z: [$ r; {; J$ ]spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced
- q! h1 ]2 Q7 @, r1 f& Cto differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups
3 [' h1 I1 [" n7 G8 _" P& @' g6 `(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve5 G* _) f+ G& x3 p9 \& d- b  J1 q
and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC2 C$ c0 k0 X* q! X' i# h# m, I  q# ?
suspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
4 w- E  L. F8 k+ R6 e) ]; Wcondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps, Z3 K' i9 J! s5 V
surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted
- M: e' w7 O" U) {for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane2 ^9 e$ B0 `$ X' {4 o1 _4 L8 @7 R
were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by8 I. R+ {8 P9 z, l( l6 Z
first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus; S, T( s: ?1 c& y" E/ ^
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) g0 x5 H3 {0 }2 X' K& Q' ~$ C
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area
" A; U2 h9 j" d4 eof the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±& F: j( R& g+ m. H# {: R
4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).1 _, p" C- J0 @% E2 y) [0 K6 J5 C
Mallory trichrome staining of muscle notified that there was more collagen fiber hyperplasia of denervated gastrocnemius in the control group than that in the experimental group at 4 and 12 weeks postoperatively. After 12 weeks of operation, the area# d9 ]; h3 n+ O2 V
of postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as+ [. |8 t4 C& K0 Z4 g$ T) M( H
(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant8 e5 d# v+ M: d9 j9 M% s
differences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of" j: B0 R1 Q& o! N% ]8 q/ j+ H5 l% G
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new- K; f5 b7 e3 O; ?
approach to prevent and treat the denervated muscle atrophy cl inically.5 L" G- b+ J1 V# ?
【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft  \2 J7 ]9 \+ T- g6 ~
Foundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and- a6 D4 P' y) g5 L- h1 }; g
Technology Commission of Shanghai Municipal ity (07JC14008)
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