2004 2-02
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  • 1. 130 Acta Physiologica Sinica, April 25, 2004, 56(2): 130-136 http://www.actaps.com.cn , , , * , 100850 : , 8d , , NF-160 , PCNA+NF-160 ( Chat GABA GAD) 8 d, , EGF bFGF 2d , ; , NF-160 , NF-160 PCNA+NF-160 ( Chat GABA GAD) , , , , : ; ; ; ; : Q254Induced-division of neurons derived from neural stem cellsLIN Qiu-Xia, QUE Hai-Ping, LU Shuang-Hong, LIU Shao-Jun*Basic Medical Institute of Academy of Military Medical Science, Beijing 100850Abstract: In order to explore if mature neurons derived from neural stem cells have the potentiality to divide we utilized the chemicaldigestion method to disperse the adult rat brain tissue into single cells, and culture them in serum-free medium. After being cultured forabout eight days in vitro, the neural stem cells were induced to differentiate into neurons. The neurons were further induced to divide. Utilizingthe method of serial photograph and NF-160 immunocytochemistry, the processes of division of some neurons were recorded. At thesame time, PCNA+NF-160 (or Chat, GABA, GAD) double label were used to investigate if the dividing-neurons were mature ones. Afterthe neural stem cells were induced to differentiate in vitro for eight days they possessed the shape and character of mature neurons. Thedifferentiated neuron had a big nucleus and one or two distinct nucleolus in the nuclear. Within the perikaryon there were a large amountof dense and Nissl body-like structure. Several long processes emerged from various locations of the cell body. Then, EGF and bFGF wereadded into the medium to induce division. After two days of induced-division, neuron-like cells were observed to divide; moreover, thenumber of neuron-like cells in the region increased continually. Immunocytochemistry demonstrated these cells were NF-160-positive.Serial photographs of dividing-process of neuron-like cells were obtained and their daughter cells were also NF-160-positive. AfterPCNA+NF-160 (or Chat, GABA, GAD) double label, some cells showed brown cell plasma and black nucleus. The above-mentioned resultsindicate that neurons, which were previously thought to be end-differentiated, can be re-called into cell cycle under appropriate conditions.Mature neurons still have the potential to divide, proliferate and self-renew.Key words: neural stem cells; mature neurons; cell division; serial photograph; immuno-double label Received 2003-06-04 Accepted 2003-07-31 This work was supported by National Basic Research Priorities Programme of China (No.001CB510206) and National NaturalScience Foundation of China (No.30140001). ∗ Corresponding author. Tel: 82-10-66931304; Fax: 82-10-68213039; E-mail: liusj@nic.bmi.ac.cn
  • 2. : 131 1.4.2 [1,2] 4d , , 4 d, [3-7] , EGF bFGF 1.4.2.1 EGF bFGF , , [8-11] , 10 d, NF-160 , : (1) 4% 15 min , 0.01 mol/L KPBS 3 10 min; (2) 5 % 30 min , NF-160 ,4 ; 0.01 mol/L KPBS 3 10 min; (3) 1:1 200 Biotinylated Goat anti-mouse IgG (0.01 mol/L1.1 Wistar , 3~4 , KPBS ), 37 30 min; 0.01 mol/L KPBS 3 10 min; (4) 1:200 Extravidin Peroxi- dase (0.01mol/L KPBS ), 37 30 min;1.2 EGF bFGF N2 B27 Gln DMEM 0.01 mol/L KPBS 3 10 min; (5) ( ) F12 Gibco ; 1 , NF-160 , ( 0.02% DAB 2%Mouse Extravidin Peroxidase staining kit DAB 0.2% 0.04% 0.0007% Sigma ; Nestin Chemicon 0.1 mol/L , pH 6.0) 20 min, ; ( ) Hyclone [12] ; (6)1.3 ( ); ( 1 , ; 0.01 mol/CO2 , Leica); SZX-9-312 ( OLYM- L KPBS 3 10 min;PUS ); CO2 (SANYO); (KUBATO) 1.4.2.2 6~7 d ,1.4 , CO21.4.1 , 20 min Wistar VZ 1 NF-160 ( SVZ, , 1mm 3 ) 2+ 2+ , ( Ca Mg 1.4.2.3 PCNA+NF-160 ( Chat , 1.33 mg/ml 0.7 mg/ml GABA GAD) 10 d 0.2 mg/ml ) DMEM/F12 , PCNA+NF-160 ( Chat GABA GAD) , 0.6%G 2 , ,mmol/L Gln 1.2 g/L NaHCO 3 20 ng/ml EGF : 1 4% 15 min ,bFGF 2%B27 1%N2 100 U/ml 0.01 mol/L KPBS 3 10 min; (2) 5 % 30 min , PCNA ,4 ; 8 d , Nestin 0.01 mol/L KPBS 3 10 min; (3)~(5) ; 1.4.2.1 (3)~(5);(6) 1 , , , ; 0.01 mol/L KPBS 3 10 EGF bFGF , min; (7) NF-160( Chat GABA GAD)
  • 3. 132 Acta Physiologica Sinica, April 25, 2004, 56(2): 130-136 4 ; 0.01 mol/L KPBS 3 10 min; 3d ,(8) (9) 1.4.2.1 (3) (4); (10) ( 1c); 8d ,( 0.02% DAB 0.2% 0.04% 0. ( 1d),0007% 0.01 mol/L KPBS, pH 7.4) , , 20 min, 0.01 mol/ ( 1e) ,L KPBS 3 10 min, ; , [13] Nestin , ,2 , 8d ,2.1 : 8 h, , , , , 1~2 , ( 1a); 12 h , , ; 24 h ( 1b), 1.Fig.1. Proliferation and differentiation of neural stem cells from the adult rat brain. Phase-contrast microscopic photographs showedthe in vitro growth, proliferation of neural stem cells isolated from adult rat brain. The cells became hypertrophic after 12 h in vitro(a) and started to divide by 1 DIV (days in vitro) (b). Arrow in b indicated the dividing neural stem cells. Proliferating cells formedsmall spheres by 3 DIV (c). Some cells continued to divide (d), the other cells began to differentiated into precursors (e, arrow) by 7~8DIV. A pair of dividing cells with typical structure and shape of neurons was shown in (f). After 50 min, the same cells were fixed by4% poly-formaldehyde and demonstrated by immunocytochemistry against NF-160. They were strongly labeled by antibody againstNF-160 (g). Scale bar = 25 µm (a,b,c), Scale bar =50 µm (d, e, f, g)
  • 4. : 133 2.Fig. 2. Induced division of neurons from neural stem cells. In each well, only a few neuron-like cells were observed at beginning. Wefound the single neuron-like cells differentiated from neural stem cells, which showed mature neurons appearance (a). We observed thecell and recorded the cell number continually. Two weeks later, the neuron-like cells in the areas increased and become a cell cluster.Immunocytochemistry showed that these cells could be labeled with NF-160 antibody (b). Scale bar =50 µm. 3.Fig. 3. Dividing neurons derived from the neural stem cells and their dividing process. A series of photographs showed the dividingcourse of two different styles of neurons (big cell: red arrow; intermediated-size cell: blue arrow) differentiated from neural stem cells.At the beginning, two neurons migrated to contact with each other (a, b); Next, perikarya became hypertrophic and lengthened (c);Following, they divided into four neurons (arrows) (d); Continually, the intermediated-size neuron formed separately two neurons. Oneprogeny from big neuron died and the other survived (e). At last, they migrated again to new places (f)~(h). The dividing course spannedabout 6 h. Immunocytochemistry showed that the three new neurons were NF-160-positive (i). Scale bar = 25 µm.
  • 5. 134 Acta Physiologica Sinica, April 25, 2004, 56(2): 130-1362.2. , 2d , ; , , , , , , NF-160 , , ( , NF-160 ,2a) , ( 3) 2.4 PCNA NF-160( Chat 14 d , GABA GAD) NF-160 PCNA , NF-160 Chat , NF-160 GABA GAD PCNA+NF- ( 2b) 160 ( Chat GABA GAD)2.3 , , : (1) , NF-160 ; ( 20 min (2) , PCNA ; (3) ) , , PCNA , ; NF-160 ( 4) 4. PCNA+NF-160 ( Chat GABA GAD)Fig. 4. Double-labeled neurons by immunocytochemistry with PCNA and NF-160 (or Chat, GABA , GAD). Double-labeled neuronshave black nuclei, brown perikarya and brown processes. The neurons shown in picture (a)~(c) were double-labeled by immunostaingswith PCNA and NF-160. The neurons shown in picture (d)~(f) were double-labeled by immunostaing with PCNA and GABA (d), PCNAand GAD (e), PCNA and Chat (f) respectively . Scale bar= 25 µm.3 19 , ,3.1 , : ,
  • 6. : 135 [1,2] , , , , , , , , , (M , ) , ; , , , , —PCNA (proliferating cell nuclear anti- , DNA , gen , ) NF-160 [14,15] , IGF IGF ( Chat GABA GAD) PCNA , , , , [17] PCNA , , , , 66~80 - - , DNA DNA ,3.2 PCNA G1 ;S ; G2 , G0 PCNA , , , , , GABA ; GAD GABA ; Chat , , NF-160 ( 2) , , , , [18] ( 30~60 min), PCNA+NF-160 ( Chat GABA GAD) , , , PCNA , NF-160 ( Chat GABA GAD) , PCNA , NF-160 ( Chat GABA GAD) NF-160 ( 3) PCNA , NF-160 ( , NF-160 Chat GABA GAD) , NF-160 , , , , , NF-H (200000) NF-M (160 000) NF-L(70 000), 3.3 : NF-L , , , , , NF-L , , NF-M NF-H , [16] , , , , NF-M NF-H , , NF-M (160000) , , ( ) ,
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