沈云东 徐建光 徐文东 徐雷 陆九州 顾玉东 m8 F' U7 r" Z' F; P【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5 x( N' I* e. a& Z" 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） / A( w2 q; W0 u9 @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$ ]% B! s$ ]0 o& 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- F: V) b' Q2 w, g
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- X8 P9 Z7 |) L+ y/ U3 T
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、# h. o ?) y2 Y4 k
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- ~/ ?3 |3 V- [+ 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 " N1 @. [8 U) z$ Y' i0 T（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 `- n% A3 D; _3 W4 ]6 @! @7 L
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% ± ( ]1 V' D& N9 J4.76% 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* Z; J ~$ }- d( f! p d) 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- |3 o4 k6 |0 e0 X8 `. o& N
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! T8 t; m& n% q
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 V) Q* O) C3 R. b$ ]- _! N4 i3 d
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& X y8 ^6 A; P0 e: p% u
的思路和方 法。8 u$ x! G0 [* ~
【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植& T2 k0 O3 e. T, ~: i
中图分类号： R651.3 Q813.1 文献标志码：A+ E) {2 I t6 O! m* f
EXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE 8 t; Q9 h' V3 b0 V" a; ^$ f Z% I+ R7 jATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,: G% z" E2 C" |$ e
Huashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@0 ]' P0 q. e7 a' ?- x
hotmail.com: J A" o0 v* T7 y [
【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated ; m2 ^& ^. N1 A7 k7 J, Y7 |muscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the# c7 d: `- n! E; K/ j8 d# i& B1 c
spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced $ e# {6 B X7 }* J( G8 {to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups% N- v x7 O/ K- O: e" x2 g
(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve; L* H) c0 Q, L. |1 Y+ | h
and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC 9 T- W9 Q( L, A. R& {* Tsuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general7 ]6 B- r, a& F. e9 N& T# E
condition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps 1 D; M9 L: n6 |6 @surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted - g1 A2 l6 {# M3 d' C0 D5 b1 Pfor the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane3 M* Z# }- {% {0 s- B1 T/ [
were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by ; A7 E# t) v2 W0 Ifirst intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus8 n! O1 u: y8 `. f$ o
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) g 6 S4 q+ Z3 w! M; jand (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area* n/ h( ?; q# Q c( ~
of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±2 t# l/ A$ K' B1 X4 k. c v! ^, J, R
4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05). 1 v% x# C; Z: `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 area4 f1 a; H4 n v/ Z% x9 e
of postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as + R* w/ [2 K9 v/ S" Y1 v; w(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant) L, y; i0 }( G7 b% U6 J
differences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of0 j# m1 b e7 l- ^2 E
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new6 e* E4 m0 h3 C U
approach to prevent and treat the denervated muscle atrophy cl inically.0 t. {" Q, o) I, ~! d* Y9 ~& C7 d* l8 l
【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft / ?1 j4 c1 g) V! i: n# C( t& ZFoundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and( E# P# a/ b" X" n
Technology Commission of Shanghai Municipal ity (07JC14008) 0 r) R# O- ^* P. Q5 ]. C+ I3 s$ M9 X' l( L" ^
附件是pdf全文。感谢大家支持，回复本贴就可以下载了 2 M0 f0 g# ^0 f- w6 s5 U1 D- V& M& d* ?% z- o1 l- M 作者: soni 时间: 2009-3-6 14:57