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沈云东 徐建光 徐文东 徐雷 陆九州 顾玉东- p4 L1 b/ R+ N
【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1 D! B: y9 t& h. i$ f7 f
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）$ A7 J- s, ^9 q* K9 @' c
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3 C! s% }1 G j% 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
( H+ q2 e1 }0 M/ E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+ ?3 S$ M3 W& M& 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、6 x% [' F) 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* b8 o) a1 ?" K- u8 v. }
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* H/ ]9 T" l" E. P
（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）。
0 Y0 {5 u2 i- h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% ±' F0 _2 h3 K2 |2 |
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
9 u- _/ }1 n2 R3 N; F- i' s4 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
: B) C8 S4 A- q( q5 l9 L3 B+ V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/ d2 Y" t: S! y: X) G" @7 w
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 X, s0 L( `/ }0 e& |
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
- ?1 R, ~' F( l" H的思路和方 法。
8 z8 z1 ]1 U3 ?" H6 e8 e【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植( S( |4 A2 _: a
中图分类号： R651.3 Q813.1 文献标志码：A
/ h$ z. @" ^6 Y5 f+ QEXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE; R9 y" z7 y+ p0 R& v
ATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,
$ L7 V- D8 N8 IHuashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@
) b" o) C; M% z, @: Ihotmail.com
, ~8 i) u3 u; R/ y/ p【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated
* m2 q4 C& _: c) [9 A$ Emuscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the
( p, d- m/ {# a' Gspinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced
: x" Z# \) z$ v, ?, R: wto differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups ]2 L1 e3 n2 S$ P8 S9 D- |' h
(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve
2 G6 w. F+ _* }& E& L1 @and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC$ C1 T, Y1 ^, f; _/ w4 R
suspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
+ ~' k4 r, _& w: e( _condition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps
/ C) ~: c: _6 _* w) nsurae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted @5 I Y5 U4 i- o3 G* P$ {% o p
for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane/ m6 c# D5 j7 Y! ?) N
were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by+ b* ]! P9 Z" y/ \; k
first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus' e& @4 _6 C1 |# ^ E3 s$ P9 C0 D
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 i! p m9 h% V t8 R4 f5 Q3 |2 P/ I
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area$ Q H @8 h) s
of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
* h3 O5 G% ~5 s4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).
! G" B( R- 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 area2 d6 r& d. Q' p% j0 l, C; d# |
of postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as# Y& s6 c0 T" k$ K3 S% E
(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant8 a7 m' J! I6 o$ f
differences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of( B h! [; g+ |8 v3 Y$ X
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new8 D. k t9 k! h* t/ R
approach to prevent and treat the denervated muscle atrophy cl inically.7 m6 B9 \; V( `# c+ {
【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft h D. j3 D2 r- O; ~6 o* i
Foundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and
, g S5 ~4 i& s2 VTechnology Commission of Shanghai Municipal ity (07JC14008)8 J( K) z$ q* O8 t& p0 g) ]4 x
5 B. n" ^* f( m+ i% z' n3 b
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发表于 2009-3-2 11:15
