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5 N: s% t- @# f
【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0 z$ A$ q. o. r+ @% o0 r7 l3 [" x
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）& r+ m% _0 \3 y0 [3 G! A: m& n
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% J" o- H- [; _, h3 D& p
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5 T2 E: q6 v$ k$ R" P" a
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5 D  v  n+ D# j, U! t8 L+ i
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、
5 g3 \* Z7 M$ ^; U$ w' TMal 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; I8 r9 ?5 U. r  `) I+ Z
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' v, F( H7 N6 V6 f) w! D9 H
（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）。' w/ u1 v; q9 Z0 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% ±
: V. l6 C% z- S8 }# _, Z$ R4.76% 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
- |: m( }  F8 s/ b  c4 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  f# ~( j& B; V: H( y
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
% e/ |, O" [; C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
' B2 `. C- r8 E4 t' C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
. Z' @& O( L: n4 G的思路和方 法。: b4 v% \3 s( @; k2 d' B. k
【关键词】 周围神经损伤 肌肉萎缩 神经干细胞 同种异体移植: i1 [- ~, P" r% C
中图分类号： R651.3 Q813.1 文献标志码：A
: y" l' m# Q! H& a' O) y. ^) w; K- JEXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE! @* U3 Y5 y- e: P
ATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,
2 `  d4 A' d1 j; [# J& B  M1 _. ?7 C) WHuashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@
; a6 a+ l: L0 ?/ p1 w* y: G$ yhotmail.com
& `5 {) K' a- T" m  e& N; y7 K! V  a【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated
% ?5 l/ U# X9 J. }) ]muscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the& ~* `8 |0 ^. i1 R/ R
spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced. ], r1 O6 z$ f, R: b3 N% Y
to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups
$ d" C+ u& q( I(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve' [4 A: E: j6 r6 h
and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC
/ i% w7 E4 t: f& U6 b5 j: l# ~( bsuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
, p9 t2 o* p+ {% s! v2 o$ H6 b$ ?+ Scondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps5 ?- a9 Q$ o' Z$ t
surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted1 \4 e  S( j' f1 M
for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane
8 x/ {1 Y3 U3 M, u! ?were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by
4 F8 u2 D# D! p; Q4 p! Y1 S* `first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus3 K$ G$ h: N' C0 o$ v
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 ~- ^1 g  z. s+ T7 d
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area; O: E) f3 c% g$ X6 `. S
of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±" c4 D  b9 L4 [7 y- T! k# T
4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).# f  R; D7 G! m% F5 ]1 @9 _( ^& @
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, {6 `  C2 X3 s1 {8 M( Hof postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as
7 C# Y7 q  ~( A, R, T  r(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant
+ a" H# d( A: |differences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of8 r. ?1 C, {8 ~/ c4 @% M' K/ m$ {8 ^
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new* M# N0 ~6 O. s2 {" B. O
approach to prevent and treat the denervated muscle atrophy cl inically.
, y  v# N& U! \1 {5 B【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft
' R% J; b7 }- B! EFoundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and
$ u# a0 P, C& _2 X) eTechnology Commission of Shanghai Municipal ity (07JC14008)
, E0 @7 `' v$ t& ~6 ]# N; B  k; Z% R" U9 R1 b- D! Y
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