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. t! K4 c, l# D/ D- k# ]4 z
【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
% O0 z: V0 e4 y; U0 ~$ O/ y* N& [( R- k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）* M( w# `# i1 |' b, R, J4 O2 }$ {
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8 l4 m1 X' Z+ s) Q7 w9 f* y
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
% W+ O  {/ e1 _5 K: X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
/ {& c4 V) {4 t, W9 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、
0 p: M. f" T7 Q  Z5 I+ rMal 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1 l% e" o) O% m$ \
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. O" P# q. C& W& v. f7 ?0 e
（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 j, s+ u5 i6 H  E/ M. ^' Y
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% ±$ \9 F7 V4 _" s
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
1 k) i9 O6 E1 {5 f4 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
) S, s* S6 ]( F6 A! c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
# W+ D* T- @& \# }8 c4 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
# l2 K% t, Z( w  f- {1 F$ X- o& Y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
: T. H4 n- h% f- N, `的思路和方 法。
2 J$ q9 X; I& F2 G6 u! I【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植
8 |2 I3 }7 }( i+ Q中图分类号： R651.3 Q813.1 文献标志码：A1 U% f- B% o! l5 q/ B
EXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE
* x+ u9 O* S, r/ c+ mATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,
6 A8 _0 ^5 c. I. D0 N* X7 [1 iHuashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@
  l3 j1 R( D7 d2 w5 ~/ U# Dhotmail.com! Y3 w6 Y5 q0 s  e" ~" ?
【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated
7 i# ]2 n5 H! I6 b) kmuscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the  L; W8 `9 ^  B: V% Q+ o9 u* H! n! \
spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced+ m! e8 M% J/ ~$ ]0 Z$ Z; F1 K  N
to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups# b% r- A! S/ I* _: ^) |1 @
(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve/ d. B# x/ l2 G4 f
and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC
. a( P& o- s9 l9 Bsuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
1 b) f* M; ]* ~( O* Ccondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps' g# y( p# S! f7 u( }* b/ v' Q1 ]" }
surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted
! }3 d6 P; y7 J0 p% f5 bfor the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane
9 F2 I- y! x# `4 ?& Y6 n4 y- Uwere detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by
9 A/ \/ ]' r$ v$ d* ~first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus
: }" M& H. h$ E3 M+ W  w) j, Ltriceps 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
& }% k( \$ A8 s, wand (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area
6 c5 R$ L  Z& gof the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
6 L* ?) T  a& x+ y5 X4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).
: B) ]( e, n" I( }& lMallory 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
2 b" `' o+ w* h  Z& N$ zof postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as9 k7 k& Q3 w+ D
(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant
- J6 k4 i% O* l" cdifferences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of
( |" k9 n! w. V( Y- l$ ^delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new
* w2 N0 T9 L: M/ U, V; F0 q% `8 G0 mapproach to prevent and treat the denervated muscle atrophy cl inically.
* p8 {- x8 c- S- w【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft9 i; i6 @9 H6 c6 _# k  I0 L2 Z; m# O
Foundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and0 t) d7 t* |% _& n& ]
Technology Commission of Shanghai Municipal ity (07JC14008): C2 d# C% {- V' v, ?. C5 x# P

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