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9 N& b9 E% G( ^& H
【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2 @$ E% m1 ?0 s9 D) {' A" }
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）2 f) C0 G6 ]6 ?( X) e! K& g
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
0 s1 w6 x7 @7 Q- ^0 q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
& g: c& V) x9 B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+ ~+ |5 m, s3 I
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、
( s! `( }3 n' u. u5 d  q4 qMal 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' U% b# y9 I, @- q
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
' w, p4 i+ C$ e2 r（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）。
: ]5 D3 O8 L6 S5 K+ D8 k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% ±
) X! c) D6 t1 I4.76% 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: }1 [, X1 d/ ?: N
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$ B, j0 s1 E3 r. `+ M: A
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$ b/ p5 n/ `) f: V8 N7 [
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6 z) [$ F9 d! e1 Q
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
$ a2 G  N, {$ P2 s的思路和方 法。, Y& k& ~% t. a) p- Z# [3 @
【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植
+ k) d$ k4 i3 N9 I中图分类号： R651.3 Q813.1 文献标志码：A
: x2 s/ l/ Y4 ]6 CEXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE
8 i: H. p0 K/ p' U9 U5 KATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,( O' k3 e1 L9 t" ?; ?
Huashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@( M, x* C  z0 Q6 K! z
hotmail.com
. \6 f5 U1 `  s6 m$ S* q【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated
* ?8 S4 ~. E; Amuscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the$ R8 z, i) @% G- Z6 r* y
spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced
) u4 C+ p0 T1 ~to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups
/ J/ Q  _- b- w9 }7 }* {(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve
/ b" L" i- Y+ n7 ?1 _and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC- l+ j0 n2 P4 v& m. v3 E, q8 s+ J/ i" ?
suspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
( w) F1 ]- c: U/ ]! ], t) lcondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps3 n% [2 L& q- M. t# x0 R- a& J7 e
surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted
# L7 J! n/ L2 S% U4 J/ ~  C( lfor the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane5 ~* h$ w: u1 ^- j6 H
were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by; Y  U% A6 |% E" ?6 f* |* `
first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus: U; S5 Q3 S( L+ a/ X
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: g* P% f0 M  L. M* w
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area; L7 {$ p: n( @5 q+ K, X
of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
- i9 p6 v+ t; q- _! e/ o4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).
+ F+ @3 a/ p3 E5 V3 z$ EMallory 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
) \4 |( Q' {% }* c& r' r4 lof postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as
$ |3 e# @5 |+ _, G8 |0 S(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant; G0 J( F0 S: g  [: M
differences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of9 P2 b% B  R8 b0 S% G/ G
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new
' d& _! g' e3 |5 c4 v3 I, {approach to prevent and treat the denervated muscle atrophy cl inically.
; g& m' g! \4 e3 o5 a2 i【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft$ v" A  I7 g; E3 ?# O$ Y: c
Foundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and6 i" A9 }( D$ K3 D
Technology Commission of Shanghai Municipal ity (07JC14008)% F$ Y+ i) S/ C% W  L; M

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