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
) q! I6 [/ E& b; b, ?. T9 r4 m【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
$ c( R$ d5 C5 z: h% J, i7 f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）
! }- H: W* N# @7 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2 N! y, s# J/ B; I2 p
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
- f% j: L- B2 H+ S! o  S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* P1 r  s, o  l3 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、
" X! p& v" n, [2 S$ F+ {+ h+ 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0 P  ^5 w& z4 q+ _  P
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 v4 _9 J: c! O% q8 M9 o; g4 o3 {! b
（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）。: L# T3 L( M, |9 j# c9 B
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% ±8 ~* [4 u4 L) R, e# m  H
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. E9 ^# J% f/ Y9 A1 I& 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 r3 N: S; m, w  i, T/ h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' T' m# t' |: x. O7 y& w2 H
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& y* C2 J0 |5 z0 k
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
" |: q9 X' t: r' _! q) }的思路和方 法。% s; c( {9 `; d: O8 j% d2 M2 i+ d
【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植
+ b9 |% Y/ N, p) w+ y中图分类号： R651.3 Q813.1 文献标志码：A$ ?) Q# ^8 N  f$ E$ v  m! K
EXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE
! W# L; R" T5 w6 T$ f7 M$ dATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,
& S( U' A' S+ c3 S2 d  o) EHuashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@
8 R7 Y% l3 }/ ]( Q/ W# ihotmail.com, n2 @- X# Y) R0 u6 \
【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated
8 ~& w& {+ ~7 bmuscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the* q# b# p' A9 f
spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced! R0 C9 q0 O4 @! Z8 S
to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups+ J7 W4 V" g$ `( @
(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve
. E# A0 \8 L2 Q4 V# h& ]  Aand commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC
, U' E( U" A& e. ^8 U* Z  M( n9 wsuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
% i: T. r$ T  s7 o. l9 scondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps
) H  S7 r& e, K6 P% c! ^% qsurae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted
) J2 Q1 H0 T, M9 ]for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane
$ F6 U: _$ ~; J. f6 w5 P) V8 [- hwere detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by& i6 ^8 R6 }6 o' O2 C+ S& _2 i* `9 Z
first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus  t0 g" l. b: O
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, [! k6 C& o8 s- k+ @5 o) T, w6 Z0 y
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area
4 G3 h3 H( H" E5 v  o( a5 _of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
6 `/ l( c4 I% E( v4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).9 L7 t% `' Z6 d* o
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
7 K$ n/ f2 V  M8 d, Z& s% b& Nof postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as/ K- C) a6 M+ Q  h3 i9 V
(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant
7 o. F. N# V5 |+ D1 Y8 qdifferences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of
; P4 ^' b: f  i- _4 zdelaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new
; C# ]  F& i0 w3 C; O2 I/ Y5 Kapproach to prevent and treat the denervated muscle atrophy cl inically.! F# J9 W6 X; k* S: Z4 K
【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft
5 K* Q4 K, g  e5 \* m& g2 T% \. p8 GFoundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and
  ~( m0 g) ?5 u; c2 V; E. HTechnology Commission of Shanghai Municipal ity (07JC14008)- r! X$ }& y' H2 y* Z) o5 ?5 M; _

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