TRPC1/ORAI1!"#$% HUVECsSOC& ROC' Ca 2+ & NO 697 andculturedtothesecond thirdpasage.genesinhuvecsweresilencedbytransfectionconstructedtrpc1or ORAI1RNA

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1 Basic& ClinicalMedicine May2017 Vol.37 No.5 : (2017) 研究论文 TRPC1/ORAI1!"#$% HUVECsSOC& ROC' Ca 2+ & NO 1,3, 1,4, 1,2, 1,2, 3, 1,4 (1 ()*+, -./0,1 2,() ; ,8 8192; 3 :;,() ;4 6 7,8 8< = >?<@, 2,AB CD )!": # 9E # F 1(TRPC1)/GHIJKG F$%L5 1(ORAI1)!"#MNO (HUVECs)G PQRG F(SOC)& #PQRG F(ROC)' Ca 2+ & NO ST $% U 2~3V HUVECs,WXY TRPC1& ORAI1 L Z[ HUVECs TJ\]^_`abc Z,real timepcr& Westernblot TRPC1 ORAI1mRNA& :L : R Z, Z (vehicle ),W 3 L CaRJ ROC TPA+CaR R X$% Calhex231 J C(PKC) Ro PKCs& PKCμ Go6967,T \ Fura 2/AM DAF FM + [Ca 2+ ] i & NO WXY TRPC1& ORAI1 Z[ HUVEC, CaRJ [Ca 2+ ] i & NO,T ] TRPC1& ORAI1 ST &' 1),TRPC1 ORAI1,mRNA& ` (P<0 05);2)M 4 8ST,TRPC1 ORAI1 Z Ca 2+ & NO \ ` (P< 0 05);3) Z TRPC1 ORAI1,] Z Ca 2+ & NO \ ` (P<0 05);4)TRPC1 ORAI1 ST!"#, M CaRJ J ST & TRPC1 ORAI1!"#] $% CaR SOC& ROCJK' Ca 2+ & NO :TRPC1;ORAI1; ;G 5;NO () :R363 *+,-:A TRPC1/ORAI1regulatesCa 2+ entryandnogenerationmediatedbyhuvecssocandroc WANGLa mei 1,3,HUQing hua 1,4,ZHONGHua 1,2,TANGNa 1,2,SUNZhi ping 3,HEFang 1,4 (1 MinistryofEducationKeyLaboratoryofXinjiangEndemicandEthnicDiseases,Shihezi832002;2 DeptofPathophysiology; 3 CentreofMedicalFunctionalExperiments,MedicalColegeofShiheziUniversity,Shihezi832002;4 Dept.ofPathophysiology, KeyLaboratoryofPulmonaryDiseaseofMinistryofHealthofChina,TongjiMedicalColege,HuazhongUniversityofScience andtechnology,wuhan430030,china) Abstract:Objective TostudythefunctionofTRPC1/ORAI1instoreandreceptor operatedca 2+ entryandnitric oxidegenerationbysocandrocinhumanumbilicalveinendothelialcels.methods HUVECswerecolected : : : ( , ) (correspondingauthor):fangf2002shz@126.com

2 TRPC1/ORAI1!"#$% HUVECsSOC& ROC' Ca 2+ & NO 697 andculturedtothesecond thirdpasage.genesinhuvecsweresilencedbytransfectionconstructedtrpc1or ORAI1RNA interferenceplasmids.theinterferenceeficiencyoftheirproteinandmrna weredeterminedby Westernblotandreal timepcr,respectively.thecelsweredividedinto:shorthairpinrna(shrna)group, controlgroupandvehiclegroup.thecelswereincubatedwithcaragonist,carnegativealostericmodulatorcal hex231andreceptor operatedchannels(roc)analoguetpa,proteinkinasec(pkc)inhibitorro , PKCsandPKCμinhibitorGo6967incubate,intracelularCa 2+ concentration([ca 2+ ] i )wasdetectedusingthe fluorescenceca 2+ indicatorfura 2/AM,theproductionofNO wasdeterminedbydaf FM ofeverygroupin HUVECs.HUVECsweretransducedwithshRNA TRPC1andshRNA ORAI1atsametime,afterculturedwith CaRagonist,[Ca 2+ ] i andtheproductionofnowasdetermined.theinteractionbetweenorai1andtrpc1were examinedbyco immunoprecipitation.results 1)shRNAtargetedtotheTRPC1orORAI1genesdecreasedtheir mrnaandprotein(p<0 05);2)Infourtreatmentgroupsundertheactionoffactors,the[Ca 2+ ] i andthenet NOfluorescenceintensityratiovaluesoftransfectionofTRPC1orORAI1shRNAgroupweresignificantlyreduced (P<0 05).3)[Ca 2+ ] i andnetnofluorescenceintensityratiooftransfectionoftrpc1andorai1shrna groupweresignificantlyreduced(p<0 05).4)ORAI1co precipitateswithtrpc1,indicatinginternationofa molecularcomplexhadenhancedbycaragonist.conclusions TRPC1,ORAI1arethecomponentsofSOCE androcechannelsinstoreandreceptor operatedca 2+ entryandnitricoxidegenerationinhumanumbilicalvein endothelialcels. Keywords:TRPC1;ORAI1;nitricoxide;Ca 2+ ;humanumbilicalveinendothelialcels G 5 (intracelularca 2+ concen tration,[ca 2+ ] i ) L5 G Ca 2+ F $, G # (Ca sensingreceptor,car)' GHI G M RG F(VOC) G P Q R G F (SOC) # P Q R G F (ROC),,SOC& ROC G F SOCJK (endoplas micreticulum,er)g ER Ca 2+, Ca 2+ / R;ROCJK G, Ca 2+ R M < CaR [Ca 2+ ] i,$% NO HI& K + F J K,!" [1] MNO (hu manumbilicalveinendothelialcels,huvecs) CaRJK ' # [Ca 2+ ] i $ / NO [2], SOC& ROC%& +' # [3], 9 E # F 1(transientre ceptorpotentialcanonical,trpc1)& GHIJKG F$%L5 1(CRACM1,ORAI1) ST ( # CaR SOC& ROC' Ca 2+ & NO 1./0$% 1 1./1 " U)*+,-./ (0O1( 6 7 7, 8F 23 45&6N78 9) ECM:; (Sciencel ); (Calbichem ); N TRPC1 #& β actin # (SantaCruze ) < N Orai1 # (Milipore );= (Protein Tech );ECL0\>?(Thermo );@ A>? real timepcr>?(takara ); Lipofectamine TM 2000 OPTI MEM & Fura 2/AM (Invitrogen );DAF FM DA(NO \ )(Be yotime );G418(Biosharp ) B C /D E >?(Omega );shrna( );FG(HI G ); J K -LLD> 1 2 "$% HUVECs :; MN:O [4] + :; HUVECs V,T Ⅷ 5 P ZQ,MN HUVECs,U RSTU

3 698 Basic&ClinicalMedicine (5) V 2~5V TW shrnaxy Z: XY, XY 70% ~90%,O Lipofectamine TM 2000(Invitrogen) PS Z LK Z, (control ) (vehicle )& R Z Real timepcr HUVECs TRPC1 ORAI1mRNA :N FG HI G " Z[ 8,O Trizol >? E RNA,\ L\\ ] ^ U _ RNA D, ` A 260nm /A 280nm M 1 8~2 0 O@ A>?W RNAa K cdna,% K b% β actinsk T re al timepcr>? mrna P PCR@c,@c #<K 25μL, SYBRPremixExTap12 5μL, PCR ForwardPrimer0 5 μl,pcr ReversePrimer 0 5μL, cdna2 0μL, 9 c K R 95 30s;PCR@c(95 5s 60 20s),406 LL< PN LL,! HUVECs TRPC1& ORAI1 : T E >? U, U 5μL TW BCA> N,% β actink 30μg U 10% SDS PAGE L,W Y (nitrocelulose,nc ) P,4 T 5% TBST 1h,L [ TR PC1 ORAI1 H #, β actin # W 4 TBST [ H = (1 5000),37 1h TBST 3 T Thermo P`Q,` N,?\ LL N HUVECs Ca 2+ : [4] N HUVECs Ca 2+ G \ < IPAsoftware P LL, n= N HUVECs NO : [4] N HUVECs NO G \ < IPA software P LL, n= ] HUVECs TRPC1 ORAI1 ST:W 2~3V HUVECs WI :;, ", G 20min [ L,12000 g 3~5min,U,[,4, O1 10# 50% ProteinGAgarose,4 10min,4 1000r/min 15min,?, ;? ProteinGAgarose, T 5, T K 500μL, &?,B,[ SDS, 5min,200 g 3min,U P SDS PAGE, J (Westernblot) 1 3 (2 T ± 5 (x±s), T SPSS17 0, T + LLLL(One wayanova), T SNK q 2 &' 2 1 shrna3456 HUVECs Z GFP shorai1, Z 0 Q \, Z RFP shtr PC1, Z 0 Q Q Z Z 48h [ G418, U 90%% R 2 2 TRPC11 ORAI1#789: U Z 48h T G418(200mg/L) P 7d HUVEC_,, Z ` (P<0 05)( 1) 2 3 HUVECs TRPC11 ORAI1mRNA#9: Z, Z mrna (P<0 05)( 2) 2 4 ; HUVECs [Ca 2+ ] i 1 NO <3#=> TRPC1 SOC& ROC ' [Ca 2+ ] i & NO ST:CaRJ ROC (12 o 13)TPA+CaR R X $% Calhex231 PKC Ro PKCs& PKCμ Go6967 8,, Z Ca 2+ & NO \ `,(P<0 05),

4 TRPC1/ORAI1!"#$% HUVECsSOC& ROC' Ca 2+ & NO 699 Ca 2+ & NO \ ` ( 3,4) ORAI1 SOC& ROC ' [Ca 2+ ] i & NO ST: ,, Z Ca 2+ & NO \ `, (P<0 05), Ca 2+ & NO \ ` ( 5,6) 2 5 TRPC1 ORAI1?@A HUVEC [Ca 2+ ] i 1 NO<3#=>, Z Ca 2+ & NO \ ` (P<0 05), Z, Z Ca 2+ & NO \ ` (P<0 05), Ca 2+ & NO \ ` ( 7,8) A relativeexpresionleveloftrpc1;b relativeexpresionleveloforail; P<0 05comparedwithcontrolgroup; # P< 0 05comparedwithvehiclegroup 1 B56C HUVECs TRPC1 Orai178#9: Fig1 ExpresionofTRPC1(A),Orai1(B)proteinexaminedbyWesternblotaftertransfectioninHUVECs(x±s,n=4) A TRPC1relativemRNAlevel;B OrailrelativemRNAlevel; P<0 05comparedwithcontrolgroup; # P<0 05com paredwithvehiclegroup 2 B56C HUVECs TRPC1 Orai1mRNA9: Fig2 ExpresionofTRPC1(A),Orai1(B)mRNAaftertransfectioninHUVECs(x±s,n=4)

5 700 Basic&ClinicalMedicine (5) P<0 05comparedwithcontrolgroup; # P<0 05comparedwithvehiclegroup 3 ; 56 TRPC1# HUVECs SOC ROCDE# Ca 2+ #=> Fig3 Efectsofthediferenttreatmentsoncalcium fluorescenceintensityinducedbysocandroc inshtrpc1 transfectedhuvecs(x±s,n=3) P<0 05comparedwithcontrolgroup; # P<0 05comparedwithvehiclegroup 4 ; 56 TRPC1# HUVECs SOC ROCDE# NO #=> Fig4 EfectsofthediferenttreatmentsonNO fluorescenceintensityinducedbysocandroc inshtrpc1 transfectedhuvecs(x±s,n=3)

6 TRPC1/ORAI1!"#$% HUVECsSOC& ROC' Ca 2+ & NO 701 P<0 05comparedwithcontrolgroup; # P<0 05comparedwithvehiclegroup 5 ; 56 Orai1# HUVECs SOC ROCDE# Ca 2+ #=> Fig5 Efectsofthediferenttreatmentsoncalcium fluorescenceintensityinducedbysocandroc inshorai1 transfectedhuvecs(x±s,n=3) P<0 05comparedwithcontrolgroup; # P<0 05comparedwithvehiclegroup 6 ; 56 Orai1# HUVECs SOC ROCDE# NO #=> Fig6 EfectsofthediferenttreatmentsonNO fluorescenceintensityinducedbysocandroc inshorai1 transfectedhuvecs(x±s,n=3)

7 702 Basic&ClinicalMedicine (5) P<0 05comparedwithshTRPC1group; # P<0 05comparedwithshOrailgroup 7 56 TRPC11 Orai1# HUVECs SOC ROCDE# Ca 2+ FG Fig7 Dynamicchangesofintracelularcalcium fluorescenceintensityratiobystoreandreceptor operatedca 2+ entryafterdiferenttreatmentsinshtrpc1andorai1 transfectedhuvecs(x±s,n=3) P<0 05comparedwithshTRPC1group; # P<0 05comparedwithshOrailgroup 8 56 TRPC11 Orai1# HUVECs SOC ROCDE# NO FG Fig8 DynamicchangesofNO fluorescenceintensitystoreandreceptor operatedca 2+ entryafterdiferent treatmentsinshtrpc1andorai1 transfectedhuvecs(x±s,n=3) 2 6 HUVEC TRPC1H ORAI1#IJ K TRPC1 #;] ORAI1 L 5, ORAI1 #;] TRPC1 L 5( 9)

8 TRPC1/ORAI1!"#$% HUVECsSOC& ROC' Ca 2+ & NO 703 P<0 05comparedwithcontrolgroup; # P<0 05comparedwithspermine+Ca 2+ group 9 ; LMN HUVECs Orai11 TRPC1IJ K Fig9 InteractionofOrai1andTRPC1proteinsafterdiferenttreatmentsinHUVECs(x±s,n=3) 3 O 0M G TRPCs W, 0# N W ER Ca 2+ STIMs& S K SOC L ORAIs 6 M TR PCs STIMs& ORAIs =!" G$ % SOCK HEK293! STIM1 ORAI1M ER PM " " JK SOC [5],M#$% TRPC1/TRPC4] SOC ;, STIM1/TRPC4 ST' SOCJK [6],M $ b TRPC6 ORAI1 STIM1 STJK SOC [7] &9E0 SOC ROC %& +' CaR ' Ca 2+ NO G F TR PC1& ORAI1 (K 'L5, % ' SOC& ROC W& / TR PC1& ORAI1shRNAL Z HUVEC,,T() HUVEC] J K SOC& ROC,0M HUVEC, TR PC1& ORAI1 SOC& ROC ' [Ca 2+ ] i $ / & NO TPA+Cal hex231 Ro ,Go6967] HUVEC,L J K SOC ROC,0 TRPC1 ORAI1* SOC % ROC, TRPC1 & ORAI1SK SOC& ROC ', # SOC& ROC ' Ca 2+ NO W TRPC1& ORAI1shRNA Z HUVEC, + Z 6,[Ca 2+ ] i $/& NO, M HUVEC TRPC1 ORAI1 ST!"#] $% CaR SOC& ROCJK ' Ca 2+ & NO 9E, ` # TRPC1& &0- ST *: [1]WestonAH,AbsiM,WardDT,etal.Evidenceinfavorof acalcium sensingreceptorin arterialendothelialcels: studieswithcalindolandcalhex231[j].circres,2005, 97: [2]./,0,1,.$ 1MO CaR' NO ST&: [J].,8

9 704 Basic&ClinicalMedicine (5) 8,2011,27: [3], $2,1,.$ RNA G # NO G & NO [J]. 83,2012,64: [4]BeridgeMJ,BootmanMD,RoderickHL,etal.Calcium signaling:dynamics,homeostasisandremodeling[j].nat Rev,2003,4: [5]YoungParkC,HooverPJ,MulinsFM,etal.STIM1clus tersandactivatescracchannelsviadirectbindingofacy tosolicdomaintoorai1[j].cel,2009,136: [6]SoursBrothersS,DingM,GrahamS,etal.Interactionbe tweentrpc1/trpc4asemblyandstim1contributesto store operatedca 2+ entryinmesangialcels[j].expbiol Med,2009,234: [7]JardinI,GómezLJ,SalidoGM,etal.Dynamicinteraction ofhtrpc6withtheorai1 STIM1complexorhTRPC3me diatesitsroleincapacitativeornon capacitativeca 2+ entry pathways[j].biochemj,2009,420: 新闻点击 PQ#RSTU 据美国 WebMD 医学新闻网 ( ) 报道, 最新研究显示, 相较于比较瘦的孩童和青少年来说, 变胖或一直很肥胖的孩童与青少年很快就会面临高血压风险增加 3 倍 这些结果尤其受到瞩目, 因为这些从过重到肥胖 或是一直很胖的孩子们在很短的时间内就罹患高血压 ; 这篇研究只持续 3 年的时间 明尼苏达州 Bloomington 的健康伙伴教育研究机构调查人员 EmilyParker 表示, 这篇研究结果强调, 开发与实施初期及有效的预防肥胖临床及公共卫生策略是很重要的 Parker 表示, 孩童与青少年有高血压是很罕见的, 我们还需要多了解高血压是否会导致这些孩子日后心血管问题风险较大 专家表示, 有高血压的孩童其肾脏功能会受损, 而且会增加 2 型糖尿病 高胆固醇与三酰甘油 以及脂肪肝的风险 对有高血压的孩子来说, 只要他们开始减重, 血压就会开始下降, 而且与高血压有关的其他疾病也会消失 这篇报告刊载在 儿科学 (Pediatrics) 在线版 V7#WXY 据美国 WebMD 医学新闻网 ( ) 报道, 最新研究指出, 曾被认为有害的鸡蛋可能并不是会令人伤心的东西 芬兰的研究人员表示, 即使是带有 apoe4 这种会增加饮食中胆固醇敏感度基因的人, 似乎都不用害怕任何鸡蛋或任何膳食中胆固醇对心脏的影响 这篇研究追踪超过 1000 位中年芬兰男性的饮食习惯 20 年, 研究人员表示, 所有人的心脏在研究开始时都是健康的, 约有 1/3 的参与者带有 apoe4 基因 芬兰 Kuopio 的东芬兰大学公共卫生与临床营养研究所的营养流行病学兼职教授 JyrkiVirtanen 表示, 大家都知道摄取膳食中的胆固醇对于血液中的胆固醇含量有适度的影响, 在大部分的研究中, 摄取胆固醇或是鸡蛋与心脏病风险较高没有关联性 Virtanen 补充说, 然而, 膳食中的胆固醇对那些带有 apoe4 基因的人的血胆固醇含量影响较大, 因此假设摄取胆固醇对这些人的心脏病风险影响较大 但这篇研究并未发现这些带有 apoe4 的人风险增加 专家表示, 根据这篇研究显示, 以健康饮食模式为前提, 每天一颗蛋似乎不会增加心脏病的风险, 或是影响膳食胆固醇 Virtanen 与同事们将研究结果刊载在 美国临床营养杂志 (AmericanJournalofClinicalNutrition)