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
* C/ H' ~( Y( ^, S. n0 \【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
$ G* W# H; j+ g9 v+ {4 E9 _* |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/ u& O( k2 H: B
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
+ X. f7 y: Z( L, k/ Z. e6 I/ @' H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0 B* R0 e* v% k% F4 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/ n9 O0 r/ ]% f; ~( t" u) X
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、3 j' ^# K5 N, [) p9 M
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
+ u; o% b" E+ w5 x( f4 o) N4 q! }3 F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6 d5 S* n3 D! k& p7 C8 H
（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/ ]7 [5 q$ v& W7 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% ±
" T* h2 Z* X1 M, u4.76% 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
7 M8 e8 F& i) k6 B( `3 V4 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' R1 q9 i* N; l5 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. R9 k+ f1 g8 v. N
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$ ?9 g1 @& c4 A, y( b  A; R
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, i1 D/ n0 Y! z2 H8 C8 R1 y
的思路和方 法。
2 ~, v4 x% s( }+ Q2 Q【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植
( z1 `8 A9 H# I7 r. [中图分类号： R651.3 Q813.1 文献标志码：A
; v. N4 Q$ F: r& C4 C5 aEXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE4 w2 A1 x+ q& l) U
ATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,
/ [/ C4 `. {6 h; ^0 g" j2 r3 wHuashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@- P7 e6 o& g1 R4 X8 M+ ]  l( |
hotmail.com
, u! B3 q0 e" D; N0 E( |【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated/ R- o6 _# S0 H6 }& U
muscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the
, q4 }  }7 F8 Nspinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced0 {; l& D' ~; X1 c: {8 g
to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups
; ?, y( I% a1 m(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve, K1 ?  b0 [- `& c! }2 Q
and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC; q& A" ~# J4 y
suspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
# L9 s( I" q* Z2 c% Q4 rcondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps: n3 Q" O& p+ v2 L5 H1 O
surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted' s7 X2 j5 ~, H. H2 y7 V6 e, E) ^) f
for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane
$ ?0 z/ z$ t) T, J" t- l) E1 xwere detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by$ ]  r$ c- Z* P7 V* z* d% X
first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus
9 y) F8 R2 D0 v3 P% {5 Htriceps 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
: @! @1 j/ [' i2 i6 T+ ^- land (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area7 O/ b! e& i7 G2 p8 t
of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
' `5 h5 n- `7 U; |; B+ L" j4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).- d7 Z( g: N; v# X% {
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
; h: `' {+ \6 Kof postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as2 c1 d% v. P( J! a. j% {
(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant; p, z3 X2 z, t, q  Y
differences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of& G2 x4 P( \% R* N* u  @
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new1 @/ M* }" s, k# J* l- {
approach to prevent and treat the denervated muscle atrophy cl inically.
0 p( E! b0 F7 S【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft" M9 D5 D6 Y: u. D- l
Foundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and
5 R; j: m' S2 i5 A) w7 M- k$ o0 FTechnology Commission of Shanghai Municipal ity (07JC14008)
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