1、do:i10. 3969/.j issn. 1006-9852. 2011. 08. 014v * *%B! y *李全波 马文庭 刘静芷 史可梅 郑宝森v(?D Sv=D.e S,?300211)K1 目的:建立一种切实可行的新生SD大鼠背根神经节神经元培养及纯化方法b方法:用显微解剖方法获取足够数量新生大鼠背根神经节,通过胰蛋白酶+EDTA消化a交替使用DF-12培养基和加有阿糖胞苷抗有丝分裂的DF-12培养基培养等方法,在体外获得纯化的背根神经节神经元,并采用NSE免疫细胞化学染色方法检测神经元的纯度b结果:获得的背根神经节神经元在体外生长良好,纯度可达到 90%以上b结论:本方法可以获
2、得大量高度纯化的大鼠背根神经节神经元b1oM 背根神经节;神经元;原代培养MODEL BULLD ING OF PR IMARY CULTURE AND PURIF ICATION OF RAT DOR SALROOT GANGLION NEURONSLiQuan-Bo,MAWen-Ting, LIU Jing-Zh,iSHIKe-Me,iZHENGBao-Sen(PainDepartmen,t SecondHospitalofTianjinMedicalUniversity, Tianjin300211)Abstract Objective: To establish a feasiblem
3、ethod ofculture and purification ofnewborndorsalrootganglionneuronsofrats. Methods: Sufficientdorsalrootganglions(DRG) wereobtainedbymicrodissec-tion, thenweredissociatedwith trypsin andEDTA. The dissociatedneuronswere culturedwithDF-12mediaandCytarabine forpurification. NSE stainingwasused todeterm
4、ine thepurificationpercentageofDRG. Results: The isolatedandculturedDRG survivedandgrew ingoodcondition invitro, andthepu-rifiedpercentageofDRGwasover90%. Conclusion: Themethod isvery feasible inobtaining themas-sivehighlypurifiednewborndorsalrootganglionneuronsofrats.K ey w ords Dorsalrootganglion;
5、 Neurons; Primary cellculturex8 $) *, *b M , * *(dorsalrootganglionneu-ron, DRG)$A, i/T20min, A, !bF 1: 200t F *+s? , 4eV, 0. 01M PBS b!3QbF 3 FtIgG, i/T20min, 0. 01M PBSb!3QbFSABC, i/T20min, 0. 01MPBS b!4Qb PDABA k4, |1mls 9 ? , 3iHq1 p9 ,1 F 3y0 ? 3b “ -v , * *% !1416d v 911, H * , Mv ,9i% , *V a8
6、% 12 14b vDDRG% !ZE8, 3i T, 324h =SD |,i/ T, Fi,i $V 3 , V P%zLC,%s zb Hc5%FBS5%HS !,iF 5ng/mlNGF, b V P%zLC, NGF V * 315bDRGc *a%a8%, 1% 8%, s N #LC y,Y * 3, L1DRGA “%8%d *%bDRG !BZES=D8 11, LCZEB# F s 05-Fu b“d *%b LCZE *%LC ,7d *%* ? aLC y+, DRG -$ Y ! !50min b“LC yd *%,BQ LC T ? b“d *%,L id *%g
7、V !Vy 9V7 *% 3; “ -ZE ! H F %s 4 d *s 9, a5- UJb L, 48hF 5Lmol/L,“; 8%,VY * 3 , P *Br90% , L (Bb ZE , DRGs V , 11, V HW = *,i P% 16, 17;N, !DF-12F i200U/mL 0. 2 g/L VrE V ? 3;O FEDTAZEh BO hz, DRG b8 , 3SDv DRG,YVAaO + EDTAha 8us DRGDF-12 !F B%ZE,v Bv * *,wb ID1 WangW, Gu J, LiYQ, eta.l Are voltage-
8、gated#496#S.eDChineseJournalofPainMedicine2011, 17, (8)sodium channels on the dorsal root ganglion in-volved inthedevelopmentofneuropathicpain?MolPain, 2011,2 XieW, StrongJA, Zhang JM, eta.l Increasedex-citability and spontaneous activity of rat sensoryneuronsfollowing invitrostimulationofsympatheti
9、cfiber sprouts in the isolated dorsal rootganglion.Pain, 2010, 151: 447459.3 SachaAM, BrianMD, DerekCM. Production ofdissociatedsensory neuron cultures and considera-tionsfortheiruse instudyingneuronalfunctionandplasticity. Nature Protocols, 2007, 153: 152160.4 Patte-MensahC, MeyerL, SchaefferVS, et
10、a.l e-lective regulation of3 alpha-hydroxysteroid oxido-reductase expression in dorsalroot ganglion neu-rons: apossiblemechanism tocopewithperipheralnerve injury-induced chronic pain. Pain, 2010,150: 522534.5 SchaefferV, MeyerL, Patte-MensahC, eta.lPro-gress indorsalrootganglionneurosteroidogenicac-
11、tivity: basicevidenceandpathophysiologicalcorre-lation. ProgNeurobio,l 2010, 92: 3341.6 Van SteenwinckelJ, NogheroA, ThibaultK, eta.l The 5-HT2A receptor ismainly expressed innociceptive sensory neurons in rat lumbar dorsalroot ganglia. Neuroscience, 2009, 161: 838846.7 BankerGA, GoslinK. Culturing
12、nerve cells. 2nded. Boston: MITPress, 1998, 545.8 k,f ,.v , * *B !.S, ,2005, 15: 591593.9 V, V,k ,.v *%B !. *%h, 2003, 11: 265267.10 f +, 3, 翀,等. 胚胎大鼠背根神经节* !B.b +D, 2006,31: 11521154.11 j, , .8Bv * * !.S=h S “5, 2009, 29: 185189.12 Sergery F, ArleenR. Protocols for neuralcellculture. 3rd ed. New Je
13、rsey: Humana Press,2001: 9597.13 LindwallC, KanjeM. The Janus of c-jun: celldeathversussurvivalandregenerationofneonatalsympathetic and sensory neurons. Exp Neuro,l2005, 196: 184194.14 LiuRY, SchmidRS, SniderWD, eta.l NGFen-hances sensory axon growth induced by lamininbutnot by the L1 cell adhesionm
14、olecule. MolCellNeurosc,i 2002, 20: 212.15 BenzviA, YagilZ, HagaliliY, eta.l Semapthor-in3A andneurotrophins: abalancebetween apo-tosis and survival signaling in embryonic DRGneurons. JNeurochem, 2006, 96: 585597.16 2U,u ,fZ.Vv 8 |*# *ZE.S1 D, 2006, 16: 367368.17 W, , . v *K“ZE.P #%h,2010, 18: 59659
15、7.5S.eD6 q) “dY 45S.eD6qIr q,4 6 ,F yq) , q HW,LT H q) f ,vTaE1 p,S.eDI X2009 M8q) “d,i2011 M b“d TLgaTLaEL ,I LI 4 ? vb g, V chttp: /www. casp. ac. cng,i g, q T?S(doc, PDFT),v1 pLgb1q a ,a) f VYV“d LCLbTgV 5, h# HI “(Te:l 010-82801712a82801705, E-mai:l casp bjmu. edu. cn;pain1712 126. com),“dVC54inyp, P“d ?zTaI4bvE5S.eD6!g!#497#S.eDChineseJournalofPainMedicine2011, 17, (8)
