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沈云东 徐建光 徐文东 徐雷 陆九州 顾玉东" Y2 p4 R9 U Z: D7 E
【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8 V- U% l+ o% \
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）: `! Q# A; P: }
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' l! B+ x( A5 q' i
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' |: g4 ]& s; ]# H+ v
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
; V2 V2 G+ |- `7 n/ |# S% c( Q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、9 i+ X5 m1 C5 }3 C
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1 s, \9 L" Q9 N0 T3 S
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 v+ K3 m i* ]7 N（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）。
' \: Q5 x6 A0 {/ M/ z) ]: v F" \7 _/ C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% ± ]: _. v. Y d6 Q7 k
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# C" [7 D7 k4 i3 v9 j
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/ [! R4 G* Q: q
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3 c1 v% w' L0 P, X# H6 v6 i F( E
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
. K ~ y3 j1 M5 c4 t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/ g1 e0 ?( v6 ?+ k2 ^3 p
的思路和方 法。
& a- R$ u0 j8 g( H7 \ 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
* c% [6 ]; f$ H% ]* y9 M! u# F3 H中图分类号： R651.3 Q813.1 文献标志码：A" r; c( m( y' _3 e7 g
EXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE
' S% A I8 I% \6 SATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,8 _" k# M0 p- ?) t! m- k
Huashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@ k8 ]# z; r* x7 b; a
hotmail.com
# t& p8 X6 c5 l/ d( d* A, S. \0 O【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated8 j" ^$ C, d8 y& M4 t! G9 Q* l
muscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the6 w1 t' X" [6 \# \$ j
spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced2 X$ h3 q3 w+ l# w. N) S
to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups
5 I& r/ U' x- x; O' H(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve, H7 ]' g4 T% l8 |( a
and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC
# U' Y, W1 ?- D) Jsuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
. j" G, ^- @" K I- {) Rcondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps- w2 u, U5 [9 |: P, e2 l
surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted( M. L( e G& x$ g; J/ S) M8 f
for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane7 w% B3 o( E8 x0 X( E9 h$ p& u
were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by ?# y2 d& p+ F! {& q3 z
first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus
4 i. Q2 B% W. T, Vtriceps 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' p3 V* U6 N1 t& G
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area
, g7 ]1 F/ @* G; Q+ ^- aof the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
( q" ?/ k: {1 O0 \4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).2 f, v6 e5 B4 }
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 area2 t% `' M A+ @% z" _
of postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as
/ }. L/ g: P. S9 N9 w(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant
7 a/ U& \/ Q, {$ j! i2 Tdifferences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of
: ^) A& L3 ~3 b% A: ?delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new( L1 L9 l1 Y$ @
approach to prevent and treat the denervated muscle atrophy cl inically.& X7 t' C4 k5 E6 S$ _
【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft
* Z/ k+ S. u& v0 L+ Y2 tFoundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and( g* G& f3 H2 r, l8 Z/ c
Technology Commission of Shanghai Municipal ity (07JC14008)
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发表于 2009-3-2 11:15
