人胎儿脊髓神经干细胞的分离培养

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刘欣春，朱悦*' L, W! b9 P$ G; _1 S. M
中国医科大学附属第一医院骨科，沈阳110001
1 y- v# \7 _4 a) {1 N( _摘要：本文旨在探讨是否能够从低温保存的流产儿分离培养出脊髓神经干细胞。将14 周流产儿在4℃下保存，2、6 和3 s- J1 b# r6 D: d7 }( Q& W
12 h 后取脊髓，将颈段、胸段、腰骶段分别进行无血清培养，并用胎牛血清诱导分化。用克隆培养的方法验证培养细# u. H- n$ \' r$ D- H
胞的干细胞特性；用免疫荧光细胞化学的方法检测神经干细胞标志nestin 及干细胞诱导分化后神经元标志MAP2、星形胶
! r- G% y4 g; J0 s+ ?质细胞标志GFAP、胆碱能标志ChAT，并比较不同时间点以及不同部位分离的神经干细胞的差异。在各个时间点，从6 J3 `! B. d% Q! U( x  K( C; k2 m
颈段、胸段、腰骶段脊髓均分离培养出具有连续增殖能力的神经球，其中腰骶段分离出的神经球数量最多，12 h 组各段, r0 W$ @" h3 D5 b! q
分离出的神经球较2、6 h 组显著减少。各段培养中的神经球均为nestin 阳性，诱导分化后均能够产生GFAP 阳性星形胶
# F* e- P. Z: f1 J) x( k质细胞、MAP2 阳性神经元以及ChAT 阳性胆碱能神经元。各段培养中的神经干细胞的克隆形成能力相似。以上结果表9 D+ [$ U+ Z) @! K. @
明，从低温保存的人胎儿能够分离培养出脊髓神经干细胞，这为基础研究以及未来治疗应用提供了新的细胞来源。1 {8 Z' N; ?: }+ i" v* A* _: a- p
关键词： 人； 流产儿； 脊髓； 神经干细胞； 培养; r2 n, P2 h# z; r
中图分类号：R338；R329.2
4 L# g8 g% ]& b" A, p. eIsolation of neural stem cells from the spinal cords of human fetus
& z7 I3 ]6 c6 K5 j% H6 MLIU Xin-Chun, ZHU Yue*$ X2 D8 |3 N9 `
Department of Orthopedics, First Affiliated Hospital, China Medical University, Shenyang 110001, China* |! e! t/ Y" D* ~4 U) L
Abstract: Neural stem cells are a potential therapeutic source for cellular transplantation therapy in neurological diseases. The present4 n- K$ g7 y% Q2 J2 F+ t8 a0 m
paper was aimed to investigate whether neural stem cells could be obtained from the spinal cords of low temperature preserved abortuses.
, t* V& T8 Z: `# q- _4 ~Fourteen weeks old abortuses were stored in a refrigerator at 4 ºC without any additional treatments for 2, 6 and 12 h before use. The
* |6 g  }7 t6 w* |& _; P* espinal cords were anatomized out and divided into cervical cords, thoracic cords and lumbar/sacral cords. Then the spinal cord segments
5 O( J' r$ v+ d+ Twere used for cell culture separately. Neural stem cells were isolated from the segments and cultured in bFGF, EGF and N2 supplement+ b# j  t1 b% n+ B* ~# H1 v( l
containing free-serum DMEM/F12 (1:1) medium. In order to examine the differentiation potential, the stem cells were induced to( D$ f) s* @4 m3 _  V9 M, j! ^
differentiate with 5% fetal bovine serum on poly-l-lysine substrate. Clonal culture was carried out to demonstrate that the isolated cells3 c! |& p! D% d0 |7 y6 n: j" R  ?
met the standard of stem cells. Indirect fluorescent immunocytochemistry was used to examine the expressions of neural stem cell; \& F5 s$ Q3 `
marker (nestin), neuron marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures
$ b! O% p( e! ?were compared. One-way analysis of variance and Kruskal-Wallis test were used for the statistical comparison. As a result, neural stem2 W( Z( E9 q, f2 r- ?4 G
cells were obtained from all the spinal cord segments with different postmortem intervals. Both the cells on the surface and inside the  `* V! y! [' p8 l4 A8 r" E
neurospheres showed nestin immunoreactivity. Therefore, nearly all the cells that composed the neurospheres were nestin-positive$ @- Q. b) j3 N  D& ]
undifferentiated cells. When the spheres were induced to differentiate, they could yield GFAP-positive astrocytes and MAP2-positive" ?5 K7 q: j$ R/ [. c
neurons including ChAT-positive cholinergic neurons. Primary neurospheres could be dissociated mechanically, expand in subcultures5 [9 S) i) j" @% G* Y4 c( _2 Y/ Z/ z
and maintain the differentiation potential. In clonal cultures, single cells from a single primary sphere could give rise to new neurospheres,
6 N/ Z/ b/ j2 Kwhich had the same differentiation potential as the primary spheres. The lumbar/sacral cord cultures gave rise to the most abundant* u* A4 x& ~( a. b2 N& F
primary neurospheres. When the preservation time of the fetus was prolonged to 12 h, the number of primary neurospheres decreased
" S7 j3 o1 B# Gsharply. The clonal formation and phenotype capacity were similar in all cultures. In conclusion, spinal neural stem cells can be isolated- b5 }- D% l1 c. C, [+ ~
from low temperature preserved abortuses and represent an alternative source for experimental and potential therapeutic purposes.
. s$ S5 l& q+ I0 FKey words: humans; aborted fetus; spinal cord; neural stem cell; culture4 `- c. P9 `! u6 \

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