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【摘要】 目的 研究叶酸对胎鼠大脑神经干细胞(NSCs)增殖的影响及其作用机制。方法 采用孕14～16 d SD胎鼠，进行NSCs原代培养。将NSCs分为4组：对照组、叶酸缺乏组、叶酸低剂量组、叶酸高剂量组。收集培养6 d的细胞，采用5-溴脱氧尿苷(BrdU)掺入法和免疫组化双标记检测NSCs的增殖情况，采用蛋白印迹法(Western blot)检测胎鼠增殖期NSCs活化的细胞外信号调节激酶1/2(pERK1/2)的表达含量。结果 叶酸低剂量组、高剂量组BrdU 巢蛋白(Nestin)阳性细胞占总细胞(70.5±2.7)，(78.8±6.9)%与对照组(64.2±8.9)%比较，差异均有统计学意义，且高剂量组明显高于低剂量组(P/ i7 w5 {! k( h9 F
【关键词】叶酸 神经干细胞 增殖 丝裂原活化蛋白激酶(MAPK)信号通路 - y# h; c* D( D$ s+ z9 C. f( o7 K0 p Effects of folate on proliferation of neural stem cells of fetal rat7 n4 N- ]- B3 H x, O& b/ E
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WANG Guanglei,HUANG Guowei,ZHANG Xumei,et al.+ y$ p8 j5 s% o& I* r3 J6 ~& `
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School of Public Health,Tianjin Medical University(Tianjin 300070,China) % N; f# c" N; S; X. d8 q' C, u! v# l6 y4 M% [& V! K/ [+ t
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Abstract： ObjectiveTo study the effect of folate on neural stem cells(NSCs) proliferation via MARK signal pathway in fetal rats in vitro.MethodsNSCs of fetal Sprague-Dawley rat from E14d to E16d were cultured,divided into four groups which were control group,folate-deficiency group,folate-low group and folate-high group.NSCs were collected after being cultrured 6 d.The proliferation of NSCs was detected by BrdU in corporating method and immunohistochemistry method.The level of pERK1/2 expression were detected by western blot method.ResultsThe percentage of Nestin and BrdU positive cells in total cells(±s) in folate-low group(70.5±2.7) and folate-high group (78.8±6.9) was significantly higher than that in control group 〔(64.2±8.9)，P 6 L1 O- y# f8 I' ]) V6 n- ] : h2 [' Q. C& ? 8 v/ I; H' P; R5 T2 x: L . f( C9 |# r& _# }5 Q* J Key words： folate;neural stem cell;proliferation;MARK signal pathway 2 j, P; l% l. D1 F3 { " _) I6 ~ {/ k5 l$ b/ g# C# n+ N 叶酸是神经系统正常发育必需的，研究证实膳实缺乏叶酸的孕妇，孕前及孕后3个月补充叶酸能够有效地预防神经管畸形的发生〔1〕。Craciunescu CN等研究发现妊娠晚期膳食缺乏叶酸影响胚胎鼠端脑部分的干细胞有丝分裂和凋亡，即能延缓细胞有丝分裂周期和降低NSCs的数量〔2〕。目前研究认为丝裂原活化蛋白激酶(mitogen-active protein kinase，MAPK)信号通路对哺乳动物中枢神经系统发育过程和调节NSCs增殖分化起重要作用〔3〕，为此，本实验采用体外培养胎鼠NSCs，探讨叶酸通过MAPK信号通路对NSCs增殖的影响。% F7 c$ q- M# L( I# l o9 g
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1材料与方法 % O, D- D6 K x& L1 Q0 N( u8 {2 b8 [2 J* e
1.1材料(1)动物：孕14～16d SD大鼠(军事医学科学院实验动物中心)。(2)试剂：IMDM(Iscove's Modified Dulbecco's Media)培养液(美国GibcoBRL公司)；碱性成纤维因子、表皮生长因子(加拿大Perotech公司)；兔抗大鼠多克隆抗体pERK1/2 IgG、小鼠抗大鼠单克隆抗体β-actin IgG(美国Santa Cruz公司)；辣根酶标记山羊抗小鼠IgG、辣根酶标记山羊抗兔IgG(北京中杉金桥公司)；化学发光试剂盒(英国Amersham公司)。(3)仪器：DYY-III8A稳压稳流定时电泳仪(北京六一仪器厂)；TS-1型脱色摇床(江苏海门其林医用仪器厂)；计算机凝胶图像分析系统(美国MLTRALUMInc公司)。% Z6 o# d) {+ {# |' B/ Z+ c
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〔2〕 Craciunescu CN,Brown EC,Mar MH，et al.Folic acid deficiency during late gestation decreases progenitor cell proliferation and increases apoptosis in fetal mouse brain［J］.Nutr,2004，134(1)：162-166.4 V: ]0 [2 G' t- L; m7 c9 l
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9 x# u/ f/ P: v# L5 q& a% C E: s 〔3〕 Jin Y，Yan Ez，Fan Y，et al.Sodium ferulate prevents amyloid-beta-induced neurotoxicity through suppression of p38 MAPK and upregulation of ERK-1/2 and Akt/proteinkinase B in rat hippocampus［J］.Acta Pharmacologica Sinica，2005，26(8)：943-951. 0 ]7 x$ W) E/ H7 q6 z , b4 @# @- O. ` 2 r1 q, h5 D7 D* V* D r6 `5 m5 D8 `3 U5 a$ t L5 P6 |/ l* {9 R
〔4〕 孙小妹，毛萌，周晖，等.脑源性神经营养因子对缺氧胚脑皮质神经元发挥保护作用的细胞内信号传导机制［J］.华西医学，2006，21(3)：454-457. * b. Y# X Y6 Z* U6 u" J7 _; P4 E3 _. ] }3 j
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〔5〕 Madan P,Calder MD,Watson AJ.Mitogen-activated protein kinase(MAPK) blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase(ERK) pathways［J］.Reproduction,2005,130(1):41-51. t% q) y& W* j8 W0 y" H& {6 K ^ , K7 u3 I/ L5 ^" Y( S5 z3 Z7 h' j % y! }( e. J9 }* @5 X! S/ z s) y4 q
〔6〕 Song HJ,Stevens CF,Gage FH.Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons［J］.Nat Neurosci,2002,5(5):438-445.1 k) L- A1 `5 j: B
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〔7〕 York RD,Yao H,Dillon T,et al.Rap1 mediates sustained MAP kinase activation induced by nerve growth factor［J］.Nature,1998,392(6676):622-626.作者: bluesuns 时间: 2015-6-1 08:18