2002-2003 > Academic research: refereed > Publication in refereed journalVersion of RecordPublishe

Chen, YY

Qian, ZM

Xiao, Q

PolyU Institutional Repository

????: 2002-04-01　????: 2002-10-08????: ????????????????? ( No.RC99038)? Th e Hong Kong Polytech nic University and Zhe-jiang Univ ersi ty Joint Supervi sion of phD Programma ( A/C:G-S966)????????:??? ( 1970- ) ,? ,?? ,??????? .????:?　? ,?? ,?? ,?? . E-mail: xiaqiang@ zjuem.zju. edu. cn大鼠心脏二价金属转运体 1表达量的不同发育期变化及其调节陈莹莹1 ,夏　强 1 ,钱忠明 2( 1.浙江大学医学院 ,浙江杭州 310031; 2.香港理工大学应用生物学与化学技术系 ,香港 )[?　? ]　目的: 研究二价金属转运体 1( diva lent me ta l transpo r ter 1, DM T1)在大鼠心脏中的表达及其受膳食铁调节的情况。方法:采用 RT-PCR和 Western blo t技术 ,观察 DM T1在雄性 Sprague-Daw ley大鼠心脏中的表达。结果:两种亚型的 DM T1 mRNA( non-IRE形式和 IRE形式 )在不同年龄 ( 7、 21、 63和 196d)的大鼠心脏中均有表达 ,随着大鼠年龄的增加 , DM T1 m RN A的表达量增加 ,并且其表达量与心脏中的铁含量呈正相关。 出生 21 d的大鼠用高铁或低铁饲料喂养 6周后 ,分别造成心脏铁超载和铁缺乏模型。 高铁组、低铁组与对照组相比 ,心脏 DM T1mRN A的表达量没有明显变化 ; Western blot分析结果显示 , DM T1蛋白 ( non-IRE形式和 IRE形式 )在铁缺乏的心脏中含量分别增加 21% 和 40% (P < 0. 01) ,在铁超载的心脏中其含量降低 26% ～ 28% (P < 0. 01)。结论: DM T1在心脏中的表达受膳食铁和心脏铁含量的调节 ,其调节方式与转铁蛋白受体不同 ,是一种转录后调节。[??? ]　铁 /代谢 ; 心 ; 载体蛋白质类 ; 二价金属转运体 1; 大鼠[????? ]　 R 541. 4　　 [????? ]　 A　　 [???? ]　 1008-9292( 2003) 02-0131-06Developmental expression and regulation of divalent metal transporter 1 in rat heartCHEN Ying-Ying1 , X IA Qiang1 , Qian Zhong-Ming2 ( 1. College of Medical Sciences, Zhejiang University ,Hangz hou 310031,China; 2.Department of Applied Biology and Chemical Technology , Hong Kong Poly technic U-niversity , Hong Kong , China )[Abstract ]　 Object ive: T o investiga te the expression of div alent metal t ranspo rte r 1 ( DM T1) m RN A in ma leSprague-Daw ley r at hear t o f different ag es and the expression o f DM T1 regula ted by dietar y iron. Methods: Re-ver se transcriptase ( RT ) -PCR and Western blot w ere used in this study . Results: ( 1) Two iso fo rms o f DM T1 mR-NA [with and without iron-responsiv e element ( IRE) ] w ere bo th detected in ra t hear t, which w ere co r related withhear t ir on content. During development , bo th of tw o iso forms o f DM T1 m RN A expression were the lowest at theage of PND 7, and inc reased at PND 21, 63 to 196. ( 2) After fed with a high iron diet or low iron diet fo r 6 w eeks,the ra ts developed iron over lo ad o r iron deficiency respectiv ely. No significant differ ences in DM T1 m RNA expres-sion w ere de tec ted among iron overload, iron deficienct and contro l rats. By using Western blo t analysis, a 21%and 40% reduction in DM T1 pro tein non-IRE fo rm and IRE fo rm respectively w ere found in iron over lo ad ra t (P <0. 01, vs contr ol) . Increases ( 26% ～ 28% ) in the lev els of two iso fo rms o f DM T1 pro tein w ere also observ ed iniron deficient rat (P < 0. 01, vs cont rol) . Conclusion: The lev el of DM T1 m RN A expression in hear t is a ge depen-dent; the tw o iso forms of DM T1 protein may be both r egula ted by ir on on the postt ransc riptional m echanism.[Key words ]　 Iron /matab; Hea rt; Ca rrier pr ot eins; Div alent m etal transpo r ter 1; Ra ts[ J Zhe jiang Univ ( Medica l Sci) , 2003, 32( 2): 131- 136. ]　　???? ,?????-??????????????????? ,??????????????????? [1 ]。 ???????? ,???????????? ( t ransferrinreceptor, T fR)?????????。?????? ,????????????????????????????? [ 2]。??????? 1( div alent metal t ranspo rter 1, DMT1)??1995?????????????????第 32卷　第 2期2003年浙 江 大 学 学 报 ( 医 学 版 )JO URN AL OF ZHE JI ANG UNIV ERSITY ( M EDICAL SCIEN CES)V ol 32　No 22003????? [3 ]。 DM T1???????? (?IRE? non-IRE) ,??? 3′-??????( unt ranslated region, U TRs)????? TfR???????? ( i ron responsiv e element ,IRE) ,????? IRE[4 ]。???? , DM T1????????????????? ,???????????? [5, 6 ]。1997? ,?????DM T1??????? [7 ] ,??? DMT1???????????????。 ??????? ,?????????????????。???????????????? DMT1m RNA?? ,?????????????????????? DM T1 mRNA???????????。1　?????1. 1　?????　?? Sprague-Daw ley???????????????。 ??????? ? ?? ? , bathophenanth ro linedisulfonicacid?? Sigma????。Taq DN A????? Gibco BRL??。 Advantage( RT-for-PCR???? Clontech????。 RNeasy Mini????? QIAGEN??。 SDS,???? ,bisacrylamide?????? Bio-Rad??。 ??、????????? PM I????。 ???? DM T1 ( non-IRE)????????DM T1( IRE)????? ADI??。1. 2　????　 A?????? 7、 21、 63、 196d??? SD?? (??????? ,? 28? ,????? 7? ) ,????????? DMT1m RNA????。 B?????? 21 d? SD?? (??? ,? 15? ) ,????? ( 25000 mg? /kg?? ,n= 5)、??? ( 60 mg? /kg?? ,n= 5)??? ( 10 mg? /kg?? ,n= 5)???? 6?? ,???? DM T1 mRNA????????????。 ?????????? ,?????????。???? , SD??? 1%????? ( 40 mg /kg , ip)??。 ?????? ,?????? ( Hb)??????。???? ,??? PBS???? ,?? ,?? ,?? [8 ]。????????????? RNA???。?????????????? [9 ]。1. 3　??? PCR ( Reverse Transcription-PCR)???? DM T1 cDN A?? ??RN easy Mini??????????? RNA。? 1μg? RNA? Advantage RT-fo r-PCR??????? cDN A。?? GenBank????? DM T1?? ( IRE? non-IRE)?? (#AF008439?# AF029757) ,????????? ?: 5′-CTGAGCGAAGAT ACCAGCG-3′;5′-GGAGCCATCACT TGACCACAC-3′? 5′-GTCT TCCAAGA TGTGGAGCA-3′, 5′-ATCT TACCCAAACTGGCACG-3′。??????? 838 bp? 750 bp???。 PCR????:94℃??? 3 min; 94℃ 45 s、 60℃ 45 s、 72℃ 2min,?? 25? ;?? 72℃?? 7 min。 β -actin?? (?? 5′-GGTCACCCACACTGT GCCCATCT A-3′; ?? 5′-GACCG TCAGGCAGCTCACATAGCTCT-3′)?????????。PCR?? 1. 8%???????? ,? Lumi-Analy st?????? ( Roche?? ) ,???????? ,? OD DM T1 /OD β-actin× 100?? DMT1??????。 PCR??? QIAEXII gel ex t raction ki t ( Qiagen?? )????? ,? ABI PrismTM 310 Genetic Ana lyzer ( PE?? )? PCR?????。1. 4　Western Blo t　?????? 1% TritonX-100 ? RIPA ? ? ? ( radioimmuno-prepeiption assay buf fer )???? , 10 000× g( 4℃ )?? 30 min,?????。? Bradford??????????。 ?? 10μg?????? 10% SDS-PAGE????? ,????PV DF?。? 1%????? ,??????????。??? 5%???????? 2 h? ,? 1∶ 5 000???? DMT1( non-IRE)???????? DM T1( IRE)??? 4℃????。TBS-T?? 3?? ,? 1∶ 5 000??????????????? 1 h。? ECL????????? DM T1??? ,?? LumiAnalyst?????? ,?????????。1. 5　????　????? x-± s?? ,??one-w ay ANOVA ? ? Student-Newman-Keuls test??????? , P < 0. 05???????。·132·　　　 ?????? (??? ) ? 32?2　?　?2. 1　 DMT1 mRNA?????????????　?? 7、 21、 63、 196 d??? SD?????? DMT1 ( non-IRE) mRNA? DMT1( IRE) mRNA???? RT-PCR?? (? 1A? 1B)。?????? ,????? DMT1???? ,? IRE??? mRNA???????non-IRE??? mRNA。??? DMT1 m R-NA,??????????? , DM T1 mRNA??????? 7 d??? ,??????。DM T1( non-IRE) mRNA?????? 196 d????? ,? DM T1( IRE) mRNA?????? 63 d???? ,??????。???? ,?? DMT1( non-IRE? IRE)???????????????? ( P < 0. 01,? 2)。? 1　?????? DM T1 mRN A???　　 (???? n= 7,? 28? )Fig. 1　 RT-PCR for detect ing th e ex pressions of tw oisoforms of th e DM T1 mRNA in rat h earts ofdi f feren t ag es (n= 7)PN D: postnatal day. * P < 0. 05,* * P < 0. 01 com-pare to non-IRE form of th e same age. + P < 0. 05, + + P< 0. 01 compare to PND7 rats. # P < 0. 05,# # P < 0. 01compare to PND21 rats.＄＄ P < 0. 01 compare to PND63rats.? 2　?? DM T1 mRNA?????????　　 ?????????Fig. 2　 Correlation betw een expressions of tw o iso-forms of the DM T1 mRN A and i ron con tent inmyocardium　2. 2　????????????????　??????? 6? ,? B??????? ,????、???? ( Hb)、????????????? (P < 0. 01,? 1)。?? ,????Hb???????? B????? ( P <0. 01,? 1) ,?????????? 6?。??? ,?? 6??????????? ,????????????????。2. 3　???????? DMT1 mRNA??????????　?? 3?? 4。 ?? 3????? ,???????????????? DMT1 mRNA???????????????。 ? Western blots???? ,?????? ,????? DMT1??????? ,?? non-IRE???????? ( 26± 9)% , IRE???????? ( 28± 8)% 。 ?? ,???non-IRE???????????? ( 79±8)% , IRE???????????? ( 60±3)% ( P < 0. 01)。Western blo ts???? ,???????????? ,?? DM T1??????? ,?? 3′-U TR?????? DM T1?????????????。 ???????DMT1???? 54 kD,???? 63 kD?? ,??? Su??????? [10 ]。??????DMT1? SDS-PAGE??????????。·133·? 2? ??? ,? .??????????? 1??????????????? 　 　? 1　高铁、低铁和对照组大鼠体重、 Hb、血清铁和心肌铁含量的变化Table 1　 Body w eight , Hb, Serum iron and hea r t non-heme ir on concentra tions in contr ol　　　　 rats ( CN ) , iron deficient r ats ( LF) and iron overload ra ts ( HF)B Group nBody weigh t( g)Hb( g /L)Serum iron( mmol /L)Heart non-h eme i ron( mg /g d ry w eigh t )CN 5 333. 00± 18. 15 178. 9± 6. 8 25. 28± 2. 54 0. 067± 0. 009LF 5 272. 14± 11. 96* * 58. 3± 8. 9* * 3. 18± 1. 20* * 0. 030± 0. 003* *HF 5 336. 43± 18. 49 195. 0± 7. 6* * 48. 31± 4. 90* * 0. 388± 0. 032* *　　* * vs cont rol P < 0. 01; * * P < 0. 01 compared wi th cont rol g roup? 3　??、???????? DM T1 mRNA　　 ?????? (n= 5)Fig. 3　 The exp ressions of two is oforms of th e DM T1mRN A in h eart s of cont rol rat s ( CN) , i ron de-ficient rat s ( LF) and iron overload rats ( HF)(n= 5)? 4　??、???????? DM T1???　　 ?????? (n= 5)Fig. 4　 Th e expres sions of tw o isoforms of the DM T1protein in hearts of con t rol rats ( CN) , i ron d e-ficien t rats ( LF) and i ron ov erload rat s ( HF)(n= 5)* * vs cont rol P < 0. 01* * P < 0. 01 compared w ith cont rol group3　?　?3. 1　 DMT1?????????????　????? ,?? DMT1?????????? ,???????????? ,?? DMT1IRE???????? DM T1 non-IRE。??????? , IRE? non-IRE????????????????。 ??? cDN A??? ,? 7? IRE??? 1? non-IRE?? ;?????? cDN A??? IRE? non-IRE???? 2∶ 4[4 ]。?? DM T1?????????????????。 ????? ,?? DM T1???????????????? ,?????????。 ??? DM T1 mRNA???????????????? ,???? DM T1???????????????。 Gunshin?????????????????? ,DM T1?? Zn2+ , M n2+ , Co2+ , Cd2+ , Cu2+ ,Ni2+ ? Pb2+ ????????? ,??????????? ; DM T1????????????? ,????????? [7 ]。?????????????????? DM T1,? Fe3+ ???????????? ( ferri reductase)??? Fe2+ [11 ]。·134·　　　 ?????? (??? ) ? 32?3. 2　?? DM T1??????????　????? DM T1 m RNA??????????。??? Caco-2??? DM T1??????????????? ,? Caco-2????????????? 5 d? , DM T1 mRNA???? , DM T1????? 2?。 mRNA????????? Caco-2?????????30%[12 ]。 ???????? ,???? DMT1??????? ,??????? DM T1??????????? [13 ]。 LMTK?? (?????????? )????????????????? 20 h? , DM T1???????? [14 ]。??????? ,?? DM T1?????????????? ,????????。?? ,? Caco-2???? ,?? DM T1??????????????。?? DMT1 mRNA?????????????? ,????DM T1??????????。???????????? (? Tf R) ,?? m RNA?? IRE,??? IRE-IRP ( ironregula to ry pro tein)???????????。???? , DM T1 ( non-IRE )?????DM T1 ( IRE)???????????。 ??? COS-7? HEK-293T ???????? [11 ]。 ??? ,?? IRE? mRNA(????????? β 1 mRNA, I???? mRNA)????????????????? [15 ] ,?????????????? (? H2O2? )???????????? [16, 17 ]。????????????? ,????? IRE-IRP???? ,?????????????? ,????? DM T1 ( non-IRE)???????。?? ,?? DM T1??????????。?????????????? DMT1?? ,??????? IRE-IRP??????????。(本课题得到香港理工大学常彦忠和柯亚博士的技术支持 ,特致谢意。)Ref erences:[1 ]　 Horwi t z L D, Rosenth al E A. Iron-mediated cardiovas-cular injury [ J] . Vasc Med , 1999, 4( 2): 93- 99.[2 ]　 Lin k G, Pinson A, Hershko C. Heart cell s in cul tu re: amodel of myocardial i ron overload and chelation [ J ]. JLab Clin Med, 1985, 106( 2): 147- 153.[ 3 ]　 Gruenheid S, Cellier M , Vidal S, et al. Iden tif icationand characteri zat ion of a second mouse Nramp gene [ J] .Genomics , 1995, 25( 2): 514- 525.[ 4 ]　 Lee P L, Gelbart T, West C, et al. The h uman Nramp2gene: ch aracteri zation of th e g ene s t ructu re, al ternativ esplicing, promoter region and polymorphisms [ J ] .Blood Cel ls Mol Dis, 1998, 24( 2): 199- 215.[ 5 ]　 Fleming M D, Trenor C C, Su M A, et al. Microcyticanaemia mice hav e a mutation in Nramp2, a candidatei ron t ransporter gene [ J ]. Nat Genet , 1997, 16( 4): 383- 386.[ 6 ]　 Garrick M D, Sco tt D, Kulju D, et al. Evidence for andconsequences of ch ronic h eme def iciency in Belg rade ratreticulocytes [ J ] . Biochim Biophys Acta, 1999, 1449( 2): 125- 136.[ 7 ]　 Gunshin H, Mack en zie B, Berger U V, et al. Cloningand characterization of a mammalian proton-coupledm etal-ion transpo rter [ J ]. Nature, 1997, 388 ( 6641):482- 488.[ 8 ]　 Zaman A K, Fu jii S, Saw a H, et al. Angio tensin-con-verting enzyme inhibi ti on at tenuates h ypofib rinolysi sand red uces cardiac perivascular fibrosis in genetical lyobese diabetic mice [ J ]. Ci rculation, 2001, 103 ( 25):3123- 3128.[9 ]　 To rrance J D, Bothw el l T H. A simple technique form easuring s torage i ron concent rations in formalinisedliv er s amples [ J] . S Af r JM ed Sci, 1968, 33( 1): 9- 11.[10 ]　 Su M A, Trenor C C, Fleming J C, et al. Th e G185Rmu tation disrup ts function of the i ron transporterNramp2 [ J] . Blood , 1998, 92( 6): 2157- 2163.[ 11 ]　 Zhang L, Lee T, Wang Y, et al. Heterolog ous expres-sion , functional characteri zation and locali zation of tw ois oforms of th e monk ey iron t ranspor ter Nram p2 [ J] .Bioch em J, 2000, 349( Pt 1): 289- 297.[ 12 ]　 Tandy S, Wil liams M, Leggett A, et al. Nramp2 ex-pression is ass ociated w i th pH-depend ent i ron uptak eacros s th e apical m emb rane of h uman intes tinal Caco-2cell s [ J] . J Biol Ch em, 2000, 275( 2): 1023- 1029.[ 13 ]　 Trind er D, Oates P S, Thomas C, et al. Localisationof divalent metal t ransporter 1 ( DM T1) to the mi-crovil lus m emb rane of rat duod enal en terocytes in i rondeficiency, b ut to hepatocytes in iron overload [ J ] .Gut , 2000, 46( 2) : 270- 276.[14 ]　 Ward rop S L, Richardson D R. The ef fect of int racel-lular iron concent ration and ni t rogen m onoxide onNramp2 ex pres sion and non-transferrin-bound iron up-tak e [ J] . Eur J Biochem, 1999, 263( 1): 41- 49.·135·? 2? ??? ,? .??????????? 1??????????????? 　 　[15 ]　 Parkes J G, Liu Y, Si rna J B, et al. Changes in geneexp ression wi th i ron loading and ch elation in cardiacmyocytes and non-myocytic fibroblas ts [ J] . JM ol CellCardiol , 2000, 32( 2): 233- 246.[16]　 Valen G, Erl W, Erikss on P, et al. Hydrogen pero-xide induces mRN A for tumou r necrosis factor alpha inh uman endo thelial cells [ J ]. Free Radic Res , 1999, 31( 6): 503- 512.[17 ]　 Nieto N , Greenw el P, Friedman S L, et al. Eth anoland arachidonic acid increase alpha 2( I) collagen ex-pression in rat h epatic s tellate cell s ov erexpressing cy-toch rom e P450 2E1. Role of H2O 2 and cycloox ygenas e-2 [ J] . J Biol Ch em, 2000, 275( 26): 20136- 20145.[责任编辑　张荣连 ]??????????????????????钱林锋 ,施丽萍 ,陈英莲(浙江大学医学院附属第一医院 ,浙江 杭州 310003)1　???????? , 80? , 70 kg,????? 20????。???+ + 。??、 CT、 MRI??:?????????、?????????。??????????????????? ,???? ,????? ,????。????? ,??3 mg /kg ,?? ACT> 480 s。?????? 24Fr??? ,?????? 32 Fr??? ,?????? 26 Fr??? ,?? Sarns 7400?????? , Terumo sx18????? ,??????。??????? ,????? 34℃?? ,?? 3. 5 L /min。???、????。???????? ,????????? ,???????????? ,?????????????????。???????????。?????? 37℃ ,????????。 ????????? 65 mmHg??。???? 1∶ 1????。?? 4 h??。?????? 1 d 60 m l,? 2 d 110 ml ,?????。??? 3 d????? ,????。2　?　??????????????? ,???????????。????????????????? ,?????????? ,????????? ,??????????????。?????? ( 1993? )????????????????????????。 ?????? ,????????????、?????、?????????。???????????????????????。 ????????????:①???????? ,????? 34℃?? ,??????????????。②?????????? ,???????????????? ,??????? ,??????????????。③???????????? (?? 50 ml /kg· min- 1 ) ,??????????? ,?????????????????????。④????? ,????????????????????? ,??????????? ,?????? ,?????????。[责任编辑　黄晓花 ]????: 2002-04-19　????: 2002-09-17????:??? ( 1976- ) ,? ,?? ,???????? .·136·　　　 ?????? (??? ) ? 32?

Developmental expression and regulation of divalent metal transporter 1 in rat heart

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