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  `8 {: y6 ^& S# W
【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
) L; Q& S% N0 w; H+ m+ v6 E, R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）
& y& {+ W( c' E" G" l/ {- ?6 J/ z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
' z8 C+ [* l- U3 x  X- @6 x1 ?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* \3 `$ d5 Q$ u. m- T. _
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+ |- ~7 t, H7 y; l/ P- W) c7 F
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、! ]" i" T4 d6 n0 S- Q
Mal 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; S2 V" |  M7 C4 X% f3 G
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
' B- v& b. s6 H/ V/ e9 A（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）。
: J4 ~9 q9 H3 }  o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% ±
/ E  a, ], v9 E4.76% 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
  B! N; f% }* [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
* {2 @' o( I/ D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9 U2 Y* r. ~' O6 l& c) ~7 H6 c0 |
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
/ O8 A. _% q+ o; H4 W. r( r& y& B3 `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0 [2 d9 E8 v* C- k
的思路和方 法。; k* |. ^/ w+ l- B% F
【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植
! c3 s1 F) }; ~# [, `3 [中图分类号： R651.3 Q813.1 文献标志码：A
* ?& V% r* f; c  oEXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE( O  @4 w4 \) R: ]! ^8 D6 U
ATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,4 G; `0 W7 B4 \# \- |0 _4 f) [
Huashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@  p1 h% _2 W) X% T0 V- g  @+ E
hotmail.com
  T6 @: y! p: O" y& P  F# F【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated7 E) t* }  f) e- \
muscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the& W4 q5 l  R3 B; r" B& b0 t
spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced
4 p8 s. O4 Q& tto differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups
- K0 [" X5 q' S0 w5 D(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve9 B# @" S. t2 V6 Y# w( J+ Q
and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC
% L4 r+ B6 z: ^2 d. asuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
) {  p% _% f/ k% v1 S1 R: I1 t7 c! kcondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps
$ t" {1 f6 ]" ]8 Csurae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted5 F; V5 i1 D) b1 g$ p1 x* T% `
for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane
* i  x* P( @- ywere detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by
0 [- c0 y( T/ P7 a; p/ v% G% x& yfirst intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus, U: M3 B- B/ b! I
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+ d1 T% ?+ g  {) E' f
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area: Q" p0 M8 |% \/ y+ M7 |) {& d
of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
& ^, i' F7 w1 F! ?) |7 D0 K4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05)." v& _, d" F0 F: d
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+ A  g( h8 i$ E) k+ |/ t+ {5 ?5 m3 f
of postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as5 m' U" z2 @/ @9 G" k7 R4 h7 ?/ E
(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant
2 P: T! s, h  ~8 v4 q" C+ fdifferences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of! Z+ F  a$ G; S
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new7 a6 p' c; a) M- z* s2 }. e
approach to prevent and treat the denervated muscle atrophy cl inically.
* ?1 W4 B! k/ ]! Y* e" ~【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft
; Y5 |; y$ Z4 ]& x3 NFoundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and; s; Z% x& g1 K
Technology Commission of Shanghai Municipal ity (07JC14008)9 {; {4 O: E9 T- j; o0 j

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