NSCs), broblast growth factor, bfgf) ( Peprotech ) ; NSCs

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1 Chinese Journal of Pathophysiology 2003,19 (3) : [ ] (2003) , 1, 2, 1, 1, 1, 1, 2 ( 1, 2, ) [ ] :( ESCs) (NSCs) : ESCs, m ller,nscs 7 d,, RT - PCR nestin glutaminase Brn - 3 nestin NSCs, : ESCs,NSCs 7 d,, nestin, BrdU, nestin glutaminase Brn - 3, : m ller ESCs, NSCs, [] ; ; [] R ; R775 [] A,, -, NSC :DMEM/ F12 ( neural stem cells, + 2 %B g/ L ( basic fi2 NSCs), broblast growth factor, bfgf) ( Peprotech ) ; NSCs :DMEM/ F %N2 + 1 %,,,(retinoic acid,ra), % NSCs, (Sigma ) Transwell (Coning ) Trizol RT - PCR ( Invitrogen ) NSCs, ( embryonic stem :- (- actin) (569 bp) ; (nestin) cells, ESCs), (327 bp) ; (glutaminase) (560 bp) ; Brain, NSCs - 3 (Brn - 3) (228 bp) ESCs NSCs, 2 1 [ ] [ ] [ ] ( 973) ( G ) ; (No ; No ) ;(No No ) : ( Tel : ; E - mail mail. com) 129 ES - D3 ( ) ; ESCs :Dulbecco s Eagle ( Gibco ) ; (embryonic body, EB) :DMEM + 20 % mmol/ L 2. 1 ESCs ESCs (1-2) 10 5 / cm 2, ESCs 24 h,48 h 1 3 [1 ] ESCs,,, ESCs (Dulbecco s modified Eagle medium, DMEM) + 20 % NSCs nestin, mmol/ L IU/ L,,,ESCs [2,3 ] 212 ESCs NSCs ESCs, / cm 2,EB EB 2 d, 1mol/ L RA,transwells

2 290 BALB/ c m ller, 2 d4 d EB / cm h,nscs NSCs 7 d 213 NSCs 7 d,10mol/ L 5 - (5 - Bromo deoxyuridine, BrdU) 24 h Thy1 NF200 MAP2 GAP43 S100 BrdU NSCs nestin [4 ] GFAP,NSCs Br2 Thy1-200 (neu2 du, rofilament protein - 200, NF200) (micro2 tubule associated protein - 2, Map2) 7 d, (growth associated protein, Gap43) S100 2 d, 2-4,, (glial fibrillary acidic protein, GFAP) 3-4 d,,, (synaptophysin), 7 d, 7 d, NSCs,, 3-5 d, 7 d, NSCs,,, 5 d nestin, nestin, NF200 MAP2 GAP43, ( retinal ganglion cells, RGCs) Thy1, S GFAP synap2 tophysin 7 d ESCs, Trizol RNA,oligodT,cDNA cdna,, PCR nestin glutaminase Brn - 3 [5-7] - actin, d BALB/ c, 1 ESCs,, 7 d, (3, ) 3 NSCs 7 d, NSCs nestin (4, ), NF200 Map2 Gap43, S100, GFAP, Thy1 (5, ) syna2 physin RT - PCR,7 d NSCs nestin glutaminase Brn - 3 (6), ESCs nestin glutaminase ESCs (1, ),(2, ), nestin 70 %, 40XY,, 3 ESCs 2 ESCs NSCs Fig 6 Positive expression of nestin, glutaminase, Brn - 3 genes in ESCs,EB NSCs by RT - PCR assays. M: DNA marker (100 bp Lad2 EB RA m ller der) ; Lane 1 : Actin ; Lane 2 : Nestin ; Lane 3 : Glutami2 4 d EB,24h, nase ; Lane 4 : Brn - 3. EB NSCs 6 RT - PCRnestin, glutaminase, Brn d,eb 2-4 d, EB,

3 291 1 ESCs NSCs ESCs NSCs, Mckay [8 ],, Okabe [9 ] ITSFn,,, 10 d NSCs,RA, ESCs,,ESCs EB,,, NSCs, B27 bfgf NSCs ESCs NSCs,,EB NSCs, [ ] 7 d NSCs,, [1 ],,,. [J ]., 2001, 17 (2) : 190 -, 192. ESCs NSCs, [2 ],,. NSCs NSCs : NSCs [J ]. ; NSCs nestin, 2001, 17 (7) : ; ; ; [3 ],,,. BALB/ c [J ]. :RGCs, 2001, 23 (1) : Brn - 3, [4 ] Lendahl U, Zimmerman LB, Macky RD. CNS stem cells ex2 -, RGCs Thy1-1990, 60 (4) : RGCs [5 ] Lee SH, Lumelsky N, Studer L, et al. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells[j ]. Nature Biotechnol, 2000, 18 (6) : ESCs NSCs [6 ] Bain G, Kitchens D, Yao M, et al. Embryonic stem cells ex2, RGCs, 168 (2) : [7 ] Mujtaba T, Piper DR, Kalyani A, et al. Lineage - restricted, neural precursors can be isolated from both the mouse neural [10,11 ] tube and cultured ES cells[j ]. Development, 1999, 214 (1) :,RGCs , RGCs, NSCs, press a new class of intermediate filament protein [J ]. Cell, press neuronal properties in vitro [J ]. Development, 1995, [8 ] Br stle O, Spiro AC, Karram K, et al. In vitro - generated, RGCs neural precursors participate in mammalian brain development [J ]. Proc Natl Acad Sci USA, 1997, 94 ( 26) : [9 ] Okabe S, Forsberg - Nilsson K, Spiro AC, et al. Develop2, ment of neuronal precursor cells and functional postmitotic, neurons from embryonic stem cells in vitro [J ]. Mech Dev,,, 1996,59 (1) : RGCs [12,13 ] ESCs [10 ] Ge J, Li YY, Zhuo YH, et al. Peripheral nerve and trans2, gene cells transplantation in the treatment of experimental ESCs NSCs, neuropathy of SD rats[j ]. Eye Sci,1998,14 (3) : , [11 ],,,.

4 292 [J ]., 2000, 21 (4s) : [ 12 ] Jian Ge, Yan Guo, Zhichong Wang, et al. Preliminary study on in vitro induced differentiation of embryonic stem cells into neurons[j ]. Eye Sci, 2000, 16 (1) : 1-6. [13 ] Li YP, Ge J, Yan J H, et al. Differentiation of embryonic stem cells into neurons and retina - like structure in nude mice[j ]. Eye Sci, 1999, 15 (3) : Preliminary study on induction of mouse embryonic stem cells into neural stem cells in vitro TAO Jing 1, GE Jian 1, HUANG Bing 2, WANG Zhi - chong 1, GUO Yan 1, YU Ke - ming 1,CHEN Hui - yi 1, CHEN Xi - gu 2 ( 1 Zhongshan Ophthalmic Center, 2 Experimental Animal Center, Sun Yat - sen University, Guangzhou , China) [ ABSTRACT] AIM :To explore the feasibility of inducing mouse embryonic stem cells into neural stem cells in vitro. METHODS : Embryonic body induced by retinoic acid and retinal m ller cells were selected in neural stem cell - defined medium for 7 days, and the morphological changes were observed. The selected cells were stained immunocyto2 chemically with anti - nestin, anti - BrdU antibodies, and their ability of expansion and differentiation were analyzed. RESUL TS :Large amounts of neurospheres were derived from embryonic body in the selected medium on the 7th day, which could be passaged and differentiated, stained positive with nestin and BrdU, and expressed nestin, glutaminase and Brn - 3 genes. CONCL USION :Neural stem cells could be derived from embryonic body induced by RA and m ller cells in the selected medium, which would offer an alternative to treat neuropathy such as glaucoma and retinal degenera2 tion in the future. [ KEY WORDS] Embryonic stem cells ; Neural regeneration ; Mice,www. bjmu. edu. cn

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