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6 h; `9 o" w( v! C& i
【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4 R* z( l+ C8 S& U, ]) ~
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）1 s5 u$ b: ^/ z# ~) L$ ~& a3 d' 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
) U; Q1 g! ]! ~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
  e& f6 O0 ]$ h- x% F. f1 N8 ^5 n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* y' o6 ^) k% z
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 F" y" Q  P/ [# \+ ]
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
! ^) Q) i% M8 u5 p  c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% r) [5 I& p* c7 n+ o0 F, S0 r& O4 v
（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）。
# a" N6 I, O7 |4 N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% ±" f6 a; w8 A' m: G$ f) J) X
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
, C4 T' _' M6 |& s% ^. b4 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
, r- V2 Z2 F0 y+ g! L, {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! Z' f! Z6 q  ?0 \
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
$ p; z& _7 S1 S3 m2 p3 A5 K3 j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
4 c5 H. E+ J4 R/ q. ^的思路和方 法。) i+ [$ `; ]8 O: A4 s7 o& T3 L
【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植) @' }4 m* U6 X' L+ l) U
中图分类号： R651.3 Q813.1 文献标志码：A4 q; L$ V: b- M# B, ?
EXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE, y5 f" w1 A& [, ?' f
ATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,
2 Y6 D% Q4 I0 R! G" hHuashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@, D- Y3 W! y9 n
hotmail.com
/ ^) ~( j1 \# N【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated
7 i" X: {" Z# R2 M9 ^/ gmuscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the
$ l7 E. T, p2 C) c; B  i0 Fspinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced: x$ Z% O' j4 N, L- m
to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups
: h/ h( f! y% w& T(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve2 a6 I( n+ `% b. h) L
and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC- j9 [6 N$ E5 H# r3 T& @! Z- w
suspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general, Z* `1 ]2 I; Q! Y6 I& M
condition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps  i+ A/ k9 J9 B. e: b, @, }
surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted  u& ?. P1 \0 W% W) b0 a
for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane+ o* S4 R4 Q# _
were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by  [( n3 q  W& O: w+ t! B( D' O3 t5 {$ p
first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus
  S! k5 c" x9 e) {4 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) g1 Q! P% x7 O7 ^
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area/ R! y# V( }& r& Q
of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
7 X. T2 l1 q/ `: T2 S4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).
# K5 K2 H% g& v0 g1 h( ]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
, P' q0 c  D  D, u% ]/ Q9 bof postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as5 e: }) X/ ?5 J: A2 _- R9 B4 E
(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant2 ~9 @5 ~9 r3 f& ]$ W4 E6 J6 X, F0 b/ U
differences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of8 [1 I2 T  w1 \% E" e4 ?
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new
& |: H5 Q  g9 o$ q0 zapproach to prevent and treat the denervated muscle atrophy cl inically.
  g, @4 b  O7 ^  e【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft! }. d6 [+ E( ]3 @& o: I& K7 s
Foundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and& j  z2 c  I. t  w+ a0 m
Technology Commission of Shanghai Municipal ity (07JC14008)
# h. T# x, x9 J* A) v/ D  p5 j. X$ X, x+ r$ K& E
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