PTX阻断溶血磷脂酸对大鼠胚胎神经干细胞的促增殖作用_英文_

PTX阻断溶血磷脂酸对大鼠胚胎神经干细胞的促增殖作用_英文_ () 文章编号 : 1007 - 6611 200801 - 0011 - 04 PTX 阻断溶血磷脂酸对大鼠胚胎神经干细胞的促增殖作用 1 2 31 2 3 () 崔慧林, 乔健天山西医科大学组胚教研室 , 太原 030001 ; 山西医科大学生理教研室 ; 通讯作者 () ( ) 摘要 : 目的 观察溶血磷脂酸 lysop hosp hatidic acid , L PA对大鼠胚胎神经干细胞 neural stem cells , N SCs的增殖的影响以 () 及百日咳毒素 pert ussis to xin , P TX对 L PA 作用的影响 。 方法 体外培养神经干细胞 ,对生成的神经干细胞球进行计数显 μ() 示增殖情况 。 结果 ?在特殊的无血清培养液中加入低浓度 L PA 0 . 01 - 1 . 0 mol/ L 后 ,神经干细胞球数量呈剂量依赖 性增加 ,表明 L PA 对 N SCs 有显著的促增殖作用 ; ?培养液中同时加入 L PA 和 P TX 后 ,神经干细胞球计数显示 L PA 引起的N SCs 的增殖被抑制了 98 % 。 结论 P TX 阻断了 L PA 对大鼠胚胎神经干细胞的促增殖作用 ,从而推测 L PA 的促增殖作用 可能主要通过 P TX 敏感的 Gi2Ras2Raf2MA P K 信号转导途径实现 。 关键词 : 溶血磷脂酸 ; 神经干细胞 ; 增殖 ; 百日咳毒素 ; 大鼠 中图分类号 : Q254 ; Q189 文献标识码 : A PTX blocks the promotive action of L PA on prol if eration of embryon ic neural stem cells in rats 1 2 3 1 ( CU I Hui2lin, Q IAO J ian2tianDept of Histology an d Em bryology , S han x i M edical U ni versi ty , T aiy u an 030001 , Chi n a ; 2 3)Dept of Physiology , S han x i M edical U ni versi t y ; Cor res pon di ng aut hor () Abstract : Object i ve To st udy t he effect of lysop hosp hatidic acid L PAo n t he p roliferatio n of rat embryo nic neural stem cells ( ) ( ) N SCsi n v i t ro and t he effect of pert ussis to xin P TXo n L PA2induced p ro motive p roliferatio n of N SCs. M et hods N SCs were μ() cult ured i n v i t ro , and neurosp heres were counted. Res ul ts ?Lower co ncent ratio ns of L PA 0 . 0121 . 0mol/ L dose2dependent2 ly increased t he numbers of neurosp heres cult ured in specific serum2f ree media , which indicated a significant p ro motive actio n of L PA μo n t he p roliferatio n of N SCs. ?Af ter N SCs were t reated by 1 . 0mol/ L L PA co mbined wit h P TX , 98 % of t he L PA2induced p rolif2eratio n was blocked. Concl usion These result s suggest t hat t he p ro motive effect L PA might be brought about mainly by t he P TX2 sensitive Gi2Ras2Raf2MA P K pat hway rat her t han t he P TX2insensitive o nes. Key words : lysop hosp hatidic acid ; neural stem cell ; p roliferatio n ; pert ussis to xin ; rat s recep to rs were mar kedly exp ressed by N SCs and L PA3 Introduction wit h L Precep to r being exp ressed f aintly by t he ( )2 A2 are operatio nally deNeural stem cells N SCs 5 fined as mitotically co mpetent , self2renewing , and same N SCs, it is interesting fo r us to o bserve t he multipotent cells able to differentiate alo ng t hree main po ssible effect of L PA o n t he p roliferatio n of embry2 ( cell are lineages in t he early neurogenesis i . e , neu2 o nic N SCs of rat s i n v i t ro and analyze t he signal 1 ,2 ) ro ns , ast rocytes , and oligo dendrocytes . Since t ransductio n pat hways as well . t he identificatio n of N SCs , t heir unique p roperties have made N SCs an at t ractive subject fo r t herapeutic Material s and methods applicatio ns to t he damaged brain . The elucidatio n of M ate ri als exter nal signals t hat might be involved in t he regula2 ( L ysop ho sp hatidic acid L PA , 12acyl2sn2glycerol232 tio n of N SCs in respect of t heir p roliferatio n and dif2 ) p ho sp hate , f at t y acid2f ree bovine serum albumin ferentiatio n may develop a replenishable so urce of re2 ( ) ( ) FA FB SA, pert ussis to xin P TX, and poly2L2ly2 ( ) placement cells. L ysop ho sp hatidic acid L PA is a sine were p urchased f ro m Sigma . DM EM/ F12 was simple p ho sp holipids mediating diverse cellular re2 f ro m Hyclo ne . B27 was f ro m Gibco2B RL . Basic fi2 spo nses , including cell p roliferatio n , t hro ugh t rans2 ( ) bro blast growt h f acto r b F GF was f ro m Pep ro 3 ,4 membrane and int racellular signaling pat hways. Tech. Mo use mo noclo nal anti2nestin was f ro m BD As we have demo nst rated in rat s recently t hat L P A1Bio science . SABC 2 cy3 kit was f ro m Bio ster . L PA ( )基金项目 : 山西省青年科技基金资助项目 20031062 () ( )at 15 - 20 D IV, t here were no at taching cells and was dissolved in p ho sp hate2buffered saline PB S co ntaining 4 % FA FB SA and sto red as stock solutio n dead cells , but neuro sp heres wit h p hase2bright sus2 () 1 mmol/ L at - 20 ?.pend in growt h medium. By immunocytochemical ex2 Pri m a ry cul t u re a n d i den t i f icat ion of N S Cs aminatio n at 20 D IV , t he cells and t he neuro sp heres were st ro ngly nestin2po sitive , identif ying t he cells are E14 rat embryo s were o btained f ro m timed2p regnant Wistar rat s wit h t he mo r ning of vaginal plug desig2 N SCs in nat ure . The p urit y of nestin2po sitive cells at ( ) 20 D IV was 96 %. nated as embryo nic day 0 E0. Cell cult ures and i2 dentificatio n were p ro duced essentially as described However , as show n in Fig 1 by neuro sp heres 5 ,6 p revio usly. co unting , w hen different co ncent ratio ns of L PA (μ) 0 . 01 - 10 mol/ L o r vehicle o nly were added into N eu ros p he res2cou n t i n g t he growt h media at 20 D IV , respectively , it t ur ned As a N SC generates a neuro sp here , co unting t he o ut t hat different co ncent ratio ns of L PA exhibited a numbers of neuro sp heres can be used to assess t he bi2directio nal o r dual actio ns o n t he p roliferatio n of p roliferatio n of N SCs. Neuro sp heres at 20 D IV were N SCs. The lower co ncent ratio ns less t han 1 . 0 dissociated by mechanical actio n , t hen t he dissociated cells were plated in uncoated 242well plates i n t he μmol/ L led to a do se2dependent increase of 2 neuro growt h medium co ntaining different co ncent ratio ns of μan in2 sp heres , it peaked at 1 . 0 mol/ L showing (μ) crease of t he numbers of neuro sp heres by up to 1 . 69 L PA 0 . 01 - 10mol/ L o r vehicle o nly. Af ter 7 d , ( ) t he numbers of neuro sp heres were co unted. To assess ?0 . 15 fold P < 0 . 01as co mpared to 79 ?3 per t he effect s of P TX , an inhibito r of Gi p rotein activa2 well in t he vehicle co nt rol ; w hile higher co ncent ra2 7 tio n, o n t he L PA2induced p roliferatio n of N SCs , μtio ns mo re t han 1 . 0 mol/ L were used , t hey caused ( ) N SCs were cot reated wit h P TX 100 ng/ ml and lower and lower p roliferatio n of cells ; and mo reover , μ1 . 0mol/ L L PA o r vehicle , respectively , and neuro2 w hen co ncent ratio ns reached and surpassed t he level sp heres co unting were made at t he same time point . μof 3 - 4 mol/ L , L PA even caused degeneratio n of The numbers of neuro sp heres were co unted under in2 N SCs as co mpared to t hat in co nt rols. Thus , 1 . 0 ( ) verted micro scope Olymp us 1M . The experiment μbe t he mo st effective do sage fo r mol/ L L PA might was repeated fo r 3 times and 6 samples were co unted p ro moting t he p roliferatio n of N SCs i n v i t ro and was f ro m each of t hem. used in t he following o bservatio ns. S t at ist ical a n al ysis The data were exp ressed as percentages of t he co nt rol ( ) values Mean ? S. E. and were submit ted to a o ne2 way ANOVA followed by Dunnet ’s po st2hoc test . Difference were co nsidered statistically significant at P < 0 . 05 . Results Ef f ects of L PA on p rol i f e rat ion of N S Cs Af ter cult uring t he co rtex of t he E14 rat embryo in growt h medium fo r 24 h , small2sized neuro sp heres Fig 1 Effect s of L PA o n t he p roliferatio n of t he rat em2 made by several cells were o bserved , wit h many dead bryo nic N SCs. Each data rep resented Mean ? S. E cells and cell debris. At 3 - 4 D IV , t he numbers of of six cult ures f ro m t hree independent experiment s neuro sp heres increased and t he size of t hem became 3 ( )P < 0 . 05 vs vehicle co nt rol larger fo r co mp rising mo re cells . Af ter t wo passages A n al ysis of si g n al i n g p at h w ays of L PA i n p rom ot2 N SCs. In t he p resent st udy , we also analyze t he po s2 sible signaling pat hways by w hich L PA brings abo ut i n g N S Cs p rol i f e rat ion it s p ro motive actio n o n p roliferatio n of N SCs. Based Fig 2 show s t hat cot reat ment wit h P TX showed no o n t he experiment s carried o ut mainly o n fibro blast s , effect in co nt rol gro up , but it almo st co mpletely 3 4 Moolenaarand van L eeuwen et alhave repo rted 2induced p roliferatio n of N SCs by re2 blocked t he L PA t hat at least fo ur G p rotein2co upled signaling pat h2 ductio n rate of 98 % as co mpared to t hat in t he co n2 ways have been identified fo r t he actio n of L PA : t wo ( ) t rol gro up t reated wit h L PA o nly P < 0 . 01. ( ) of t hem are P TX2sensitive t hro ugh Gi p roteinand ( t he ot her t wo are P TX2insensitive t hro ugh Gq p ro2 ) tein. In o ur p resent st udy , P TX has blocked up mo st of t he L PA2induced p roliferatio n of N SCs , and t hus excludes t he po ssibilit y t hat P TX2insensitive sig2 naling pat hways might play a rat her impo rtant role in t he blocking p rocesses to a visible extent . On t he ot h2 er hand , it is generally accep ted t hat o ut of t wo P TX2 sensitive pat hways , o ne is to inhibit t he membrane 3 effecto r adenylyl cyclaseand t he ot her is to activate Ras p rotein and t hen t he dow nst ream Raf / MA P K Fig 2 Effect s of P TX o n t he L PA2induced p roliferatio n of 9 cascade to stimulate t he cell p roliferatio n. Indeed , 3 ( )rat embryo nic N SCs P < 0 . 05 vs L PA alo ne so me repo rt showed t hat L PA did induce a decrease of cAM P t hro ugh Gi p rotein and t hen t he inhibitio n of Discussion 10 adenylyl cyclase . However , as indicated recently Our result s have show n t hat t he lower co ncent ratio ns 11 by Kranenburg and Moolenaar , acco rding to t he of L PA can p ro mote t he p roliferatio n of N SCs , wit h data o btained mainly f ro m t he p roliferatio n of quies2 μt he co ncent ratio n of 1 . 0 mol/ L being t he best fo r cent fibro blast s , Ras activatio n co uld be f ully inhibit2 t his effect ; and w hen t he do sage increases co ntinu2 ed by P TX , yet not seco ndary to Gi2mediated inhibi2 μo usly and surpasses up to 3 . 0 mol/ L , L PA exhibit s tio n of adenylyl cyclase . Thus , it is reaso nable to degenerative actio n upo n t he cult ured N SCs as co m2 p ropo se t hat t he p ro motive actio n of L PA o n p rolifer2 pared to t he blank co nt rol , and even causes total atio n of N SCs might be almo st co mpletely bro ught μdeat h of cells o nce 10mol/ L o r mo re L PA have been abo ut t hro ugh t he Gi2Ras2Raf / MA P K signaling pat h2 reached. These result s were in co nsistent wit h t hat way , leaving a few roo m fo r t he participatio n of Gi2 o btained in o ur p revio us st udies o n t he effect s of L PA AC2cAM P cascade and ot her pat hways into t he p ro2 upo n t he p roliferatio n of rat embryo nic neural stem motive p rocesses. The questio ns t hat need to answer 3 6 cells by H 2t hymidine inco rpo ratio n. Co nto s et remain : we still do not know w hat are t he membrane 8 al has repo rted t hat co2incubatio n wit h 100 nmol/ L effecto r and t he seco nd messenger in t he plausible Gi2 of L PA increases t he p roliferatio n by 25 % in cult ured Ras2Raf / MA P K signaling pat hway co ncer ning t he neuro blast s taken f ro m E12 - 13 mo use embryo nic L PA2induced cell p roliferatio n , and w hy Gi p rotein co rtices following 18 h expo sure to L PA . So L PA can ( activates here rat her t han“inhibit s”as it generally be t ho ught as a f avo urite candidate fo r p ro moting t he ) doest he Ras and dow nst ream subst rates in t he sig2 p roliferatio n of N SCs. naling pat hway t hat we are interested in . These 5 Our recent repo rt showed t hat L Pand L P A1 A3p ro blems await f urt her investigatio n . In summary , recep to rs were mar kedly exp ressed by N SCs wit h o ur result s have show n t hat L PA at it s lower co ncen2 L Precep to r being exp ressed f aintly by t he same A2 t ratio ns st ro ngly p ro motes t he p roliferatio n of N SCs rentiatio n to cholinergic neuro ns i n v i t ro J . Shen gli Xuebao , by activating t he Gi2Ras2Raf2MA P K signaling pat h2 2006 ,58 :547 - 555 . way to stimulate t he mitogenic activit y in t hese cells van Co rven EJ , Ho rdijk PL ,Medema R H , et al . Pert ussis to xin2 7 t hro ugh L Pand L Precep to rs. A1 A3 sensitive activatio n of p21 ras by G p rotein2co upled recepto r Ref erences ago nist s in fibro blast s J . Proc Natl Acad Sci U SA , 1993 , 90 : 1 2 Seaberg RM ,van der Kooy D. Stem and p rogenito r cells :t he p re1257 - 1261 . mat ure desertio n of rigo ro us definitio ns J . Trends Neuro sci , Co nto s JJ , Fukushima N , Weiner J A , et al . Requirement fo r t he 8 2003 ,26 :125 - 131 . lpA1 lysop ho sp hatidic acid recepto r gene in no r mal suckling be2 2 Gage F H , Ray J , Fisher LJ . Isolatio n ,characterizatio n ,and use of havio r J . Proc Natl Acad Sci U SA ,2000 ,97 :13384 - 13389 . stem cells f ro m t he CNSJ . Annu Rev Neuro sci ,1995 ,18 : 159 9 An S ,Bleu T , Zheng Y , et al . Reco mbinant human G p rotein2 - 192 . co upled lysop ho sp hatidic acid recepto rs mediate int racellular cal2 3 Moolenaar WH. L ysop ho sp hatidic acid , a multif unctio nal p ho s2 cium mo bilizatio n J . Mol Phar macol ,1998 ,54 :881 - 888 . p holipid messenger J . J Biol Chem ,1995 ,270 :12949 - 12952 . 10 van Co rven EJ , Groenink A ,J alink K , et al . L ysop ho sp hatidate2 4 van L eeuwen FN , Giep mans BN ,van Meeteren L A , et al . L ys2 induced cell p roliferatio n :identificatio n and dissectio n of signaling op ho sp hatidic acid : mitogen and motilit y facto r J . Biochem Soc pat hways mediated by G p roteinsJ . Cell ,1989 ,59 :45 - 54 . Trans ,2003 ,31 :1209 - 1212 . Kranenburg O ,Moolenaar WH. Ras2MA P kinase signaling by ly2 11 5 Cui HL , Qiao J T. Effect of lysop ho sp hatidic acid o n differentia2 sop ho sp hatidic acid and ot her G p rotein2co upled recepto r ago nist s tio n of neuroglia f ro m rat embryo nic neural stem cells i n v i t ro J . Onco gene ,2001 ,20 :1540 - 1546 . J . Shen gli Xuebao ,2007 :in p ress. 作者简介 : 崔慧林 ,女 ,1971 - 01 生 ,博士 ,副教授 . [ 收稿日期 : 2007 - 09 - 14 6 Cui HL , Qiao J T. Pro motive actio n of lysop ho sp hatidic acid o n p roliferatio n of rat embryo nic neural stem cells and t heir diffe2 致谢 近年《山西医科大学学报》陆续被《CA》、俄罗斯《文摘杂志》等收录 ,这与长期支持和帮助编辑部把关的 () 各位评审和专家分不开 ,在此致以衷心感谢 。以下是 2007 年为本刊审稿的专家 按汉语拼音排序:白 莉 白小红 毕小平 陈还珍 成霄黎 褚银平 邓志华 丁 红 杜永成 段虎斌 郭 政 郭东星 郭瑜玲 郝解贺 霍丽娟 韩冲芳 韩清华 韩树峰 姜 峰 焦艳军 晋建华 景 雅 康春松 李 莉 李 琳 李 兴 李灵敏 李明娥 李佩珍 李青山 刘 强 李荣山 李思进 李天亮 李卫星 李祥生 李昭英 刘保江 刘慧荣 刘桂芬 梁景青 刘近春 刘立新 刘秀梅 刘学军 刘玉玺 梁建芳 罗晓晋 马 宏 南欣荣 田秀珠 倪淑华 牛 侨 裴秋玲 彭 清 曲成毅 孙克勤 王 彤 王 彦 王春芳 王东文 王桂琴 王明正 王小琴 王英元 吴博威 武明虎 武希润 解 军 吴素慧 张 策 张 辉 张爱平 杨桂娇 杨官娥 杨 红 杨林花 阴怀清 殷国荣 张宝林 张唤平 张克让 张明升 张淑秋 张伟华 厶 张秀莲 张岩波 张雁钢 克明 贝 赵长青 赵浩亮 赵龙凤 赵振林 甄 丽 郑绘霞 张新日 郑建中 本刊编辑部