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+ `8 c, z$ M4 ^/ v
【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 |& r, v: T$ u# F3 E. }) r: 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）
- b5 B$ u5 T* f5 K2 m) @: }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
- F7 M0 Y! J* ?8 c1 |* 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
. M# l4 ^& ?& t$ j0 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
9 Q+ U1 g! k5 A  B  x! f( C) j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 @" j, D* c% p9 |, J2 XMal 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
. ^. M# v; |+ m! ?! X* o, Q/ H+ t2 b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
2 N% W8 U  k+ g; C3 [$ {5 u（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）。/ j4 f7 T; C0 G& D& 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% ±( \0 u, o& e5 ]
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7 {" T9 P1 f, G3 P4 ?( i
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$ L' W/ j. p$ `1 Y3 u2 e4 |
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0 S& {* T+ u4 E+ Y
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5 T& c+ W1 Q; S4 G; ], P4 m
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
( d$ z# A0 }8 `- f6 h$ ]8 V: W的思路和方 法。6 [/ C% a# D; m/ 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
4 X' Q+ s' I  I9 F. h7 |# O中图分类号： R651.3 Q813.1 文献标志码：A
3 I4 Z0 A; e" X4 `4 `& L: X: L, y, JEXPERIMENTAL STUDY ON NEURAL STEM CELL TRANSPLANTATION DELAYING DENERVATED MUSCLE0 |9 Y% C) ~" R* e
ATROPHY/SHEN Yundong, XU Jianguang, XU Wendong, XU Lei, LU Jiuzhou, GU Yudong. Department of Hand Surgery,
/ W$ H7 @" K- `Huashan Hospital, Fudan University, Shanghai, 200040, P.R.China. Corresponding author: XU Jianguang, E-mail: xujianguang@
8 P7 [  n4 P' W, Ehotmail.com
+ g5 u) r0 R9 D& Y$ B+ @【Abstract】 Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated! M- ]6 E9 t/ X8 G/ I
muscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the+ a* w8 |3 U8 o. R. W
spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced
6 `5 ]2 z, x/ a8 L* G- S5 [& Vto differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups9 d9 c7 G2 [1 q. f8 }
(n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve
; F' E2 ~! T- p; a! n5 k8 ~& @! gand commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSC
/ x( Q! c* j: e$ Jsuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectively. The general
( u4 e1 Z, H0 F! e. Z( hcondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps
/ {2 z3 S4 x4 I2 }surae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted
# ]5 ]2 }7 V5 B2 M! ^) c% Y* vfor the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane) m) ?5 H. E: N8 h7 O
were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by
6 R( h; _6 ~3 t& {5 wfirst intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus/ N8 u; @! R' I  b: b7 C; 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, O3 U9 Q& H& d/ A& i  u. i' ?
and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P < 0.05). The fiber section area
6 l7 R, t- Y. O- n1 j7 i1 i+ wof the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ±
; L4 Z+ b" k0 i9 f$ X: x4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P < 0.05).2 i6 r' ]% Q, S4 i+ ]' n
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
# N. X4 W% a! x# v4 hof postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as% Y& Y3 T# d, a3 l4 q& m
(61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant. R3 w% y8 P$ p
differences (P < 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of3 j# w! u1 n3 J, L
delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new1 ~# L8 w2 f7 f; E' m0 q6 E1 ]+ W
approach to prevent and treat the denervated muscle atrophy cl inically.8 P9 ?" ?9 {, `" w1 i. p$ E
【Key words】 Peripheral nerve injury Muscle atrophy Neural stem cell Allograft
  A4 x( A/ c0 ?7 |# HFoundation items: National Natural Science Foundation of China (30672124); Natural Science Foundation of Science and; g' X- @; _) K. V! O
Technology Commission of Shanghai Municipal ity (07JC14008)
5 l) H2 H/ [+ s
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