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
# s6 M) N: A. l0 \2 D; c. w$ _% w7 ^) v【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; k& I# s8 i  t3 a- Z  m
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）3 r1 c. D1 o/ V0 p, M
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
7 B( O, \6 d# ]3 p8 N( H7 `- e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
% v/ Y# E' B4 d/ r3 m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! A/ ?. Z) V" o3 G" Q- |4 x. P
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、
2 d' k) p# o" 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1 \% J) {8 `. D+ T( z6 o
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/ K* h$ U, O$ ^, m9 M9 s1 w: 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+ s5 P/ J' F1 l# G6 N% j
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% ±- S7 r  V9 ~: P) p: F
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. Y! b6 N! c5 F0 {/ K6 k
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) M. \1 O9 x) i) S% [) ~5 E) E/ c1 Z
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, P2 Q' p) ~" m9 `3 O
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
1 M# T; ^- o! X0 d$ a) h4 ~. j2 ]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: p: ]2 |$ M1 w* t5 [5 y
的思路和方 法。+ Q! Q. \# s9 a$ w6 P3 v
【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植
% T/ d& J! Z- P8 M  y+ Z; ?1 o7 t中图分类号： R651.3 Q813.1 文献标志码：A$ V- C: w2 H2 |& c3 v- i
EXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE; g5 `& S: Z4 W' H
ATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,
8 k5 ]9 h( }) J5 OHuashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@
/ u  u) Q6 v1 t5 @3 {hotmail.com; t% I, \' ^* ^% R  b
【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated
- C" C' d& d$ Fmuscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the3 \0 F: M( j" \. n% H" I: h' @+ W
spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced
' A% K' V! ~, K* Xto differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups
6 W9 N, s' I9 [: }# k% ~(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve
. }3 J4 h& X. c3 [' w5 Kand commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC
! b5 k8 g0 G1 y, Rsuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general7 v3 l; T# l3 H" h1 F- M% R4 }+ {
condition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps
  G2 b3 {* l. u" h$ H7 gsurae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted
& v0 \; l8 P9 j& f+ O1 |: i" K' v# J  Wfor the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane5 Z( i, w- @, |  L: Q7 e
were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by9 H8 d$ f; M. O: n- v
first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus
5 B% X3 @, T: ^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) g5 t+ b- V, Q" J& H2 s* H2 w. r
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area
( _) S3 e8 N# H& ?+ Pof the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
, V% ^7 q3 `+ G4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).7 i8 {$ i- J. o# G; |. a% E# `
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
' ?5 A4 d  o8 N4 z, fof postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as! x2 ~' v9 f! Z1 @* ^4 V
(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant/ P7 ^4 R: d* F! S4 E
differences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of# g3 s/ Q1 ]8 t1 A9 d# _( _; D
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new$ M2 ^% k/ i+ p) c% @* x+ t! {( n2 ]( o
approach to prevent and treat the denervated muscle atrophy cl inically.
& |8 Y/ b" n0 N9 M% r7 _【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft
- [; ?" U: u7 K( k5 n: U5 yFoundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and
5 P5 _+ g* E' J0 X7 P! fTechnology Commission of Shanghai Municipal ity (07JC14008)) g& Z! T$ b/ a9 g1 G
. x4 ~3 [6 d; E1 `( t3 T
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