:TGF-β bfgf 23 KEY WORDS:transforminggrowthfactor-β ;basicfibroblastgrowthfactor; bonemarrow mesenchymalstemcels;eukaryoticexpressionvector; liposomet

Size: px

Start display at page:

Download ":TGF-β bfgf 23 KEY WORDS:transforminggrowthfactor-β ;basicfibroblastgrowthfactor; bonemarrow mesenchymalstemcels;eukaryoticexpressionvector; liposomet"

豚窑米
5 years ago
Views:

1 2 ( ) JournalofNanchangUniversity(MedicalSciences)208,Vol.58No. TGF-β bfgf,, 2 (., ; 2., 5040) : -β (TGF-β ) (bfgf) (BMSCs), SD BMSCs Percol PCR TGF-β bfgf, pcdna3.(+), 3 BMSCs RT-PCR Westernblot BMSCs bfgf TGFβ Percol BMSCs pcdna3.(+)-bfgf pcdna3.(+)-tgf-β, BMSCs,RT-PCR Westernblot bfgf TGF-β bfgf TGF-β BMSCs, : -β ; ; ; : R78 ;Q78 : A : (208) DOI:0.3764/j.cnki.ncdm BoneMarrow MesenchymalStem CelsModified bytgf-βandbfgf DAIFang,SONGLi,ZHU De-xing 2,ZOUYuan (.Departmentof Stomatology,theSecond Affiliated Hospitalof Nanchang University,Nanchang330006,China;2.Departmentof Oral Medicine, Stomatology Hospitalof Guangzhou Medical University,Guangzhou5040,China) ABSTRACT:Objective To modifybone marrow mesenchymalstem cels(bmscs)usinglipo- some-mediatedtransforminggrowthfactor-β (TGF-β )andbasicfibroblastgrowthfactor(bfgf) genes,andtoobservetheexpressionofthetwogenes.methods TheBMSCswereisolatedfrom SDratsbyadherencemethodcombinedwithPercoldensitygradientcentrifugation.TheTGF-β andbfgfgeneswereobtainedbypcrtoconstructtheeukaryoticexpressionvectorpcdna3. (+).ThesetwovectorsweretransfectedintothethirdgenerationofBMSCsusingtheliposome. TheexpressionofbFGFandTGF-β inbmscswasdetectedbyrt-pcrand Westernblot.Re- sults TheBMSCswereobtainedbyadherence methodcombined withpercoldensitygradient centrifugation.aftertransfectionwiththeconstructedpcdna3.(+)-bfgfandpcdna3.(+)- TGF-β eukaryoticexpressionvectors,theexpressionofbfgfgeneandtgf-β wasdetectedin BMSCsbybothRT-PCRand Westernblot.Conclusion ThebFGFandTGF-β canbehighlyex- pressedinbmscsmodifiedbyliposometransfection. : : (200BSA5900) : (983 ),,,, :,, song @26.com

2 :TGF-β bfgf 23 KEY WORDS:transforminggrowthfactor-β ;basicfibroblastgrowthfactor; bonemarrow mesenchymalstemcels;eukaryoticexpressionvector; liposometransfection ( ), IPTG X-Gal, ;, ;CD29 CD90FITC, BioLegend ;bfgf TGFβ CD34PE β -actin, SantaCruz ;CD45PE, ebioscience ;TRIZOL Opti-MEM Medium [-2] (MSCs) Lipofectamine2000 Invitrogen ;,,, :WD-9403; [3],, :HH-W2-600;CO 2, :30;, BectonDickinson,FACSCali-, bur;,, [4] :XDS-B -β (TGF-β ).2 (BMSCs) 4 SPF SD 20, [5-6] 60~00g, (bfgf) MSCs ;,,, ;, [7-8], TGF-β bfgf BM-.3 SCs,,.3.. pcdna3.(+)(5427bp) pmd20-tvector E.coliDH5α ATC AAGCTT ACCATGCCGCCCTCCGGGCT - ;RT-PCR GCGGCTGCTG-3,R 5 -ATCCTCGAGTCAG- PrimeScriptTMstStrandcDNA Synthe- CTGCACTTGCAGGAGCGCAC-3 (468bp);bFG- sis DNA Marker TaKaRa F:F 5 -ATC AAGCTT ACCATGGCAGCCGG- ; PCR GAGCATCACCACGCTG-3,R 5 -ATCCTCGAG- TTAGCTCTTAGCAGACATTGGCAG -3 (73bp) ;T4 Hind Ⅲ XhoⅠ.3.2 RNA RT-PCR Therom Fisher ; F2/ DMEM Hyclone ; NCBI TGF-β bfgf mrna (NC ,NC ), Primer5.0 HindⅢ XhoⅠ ( ) TGF-β :F 5 - RNA Trizol,,

3 24-80, PBS,20 400r min - 0 min, RNA, PCR,, cm - PCR :63.6 5s,98 0s,72 25cm 2, 0 ml,37 min, 30 tgf.fgfb 5%CO 2 24h, % 24h BMSCs, /.3.3 TGF-β bfgf PCR μl pmd20-tvector tgf.fgfb, 3μL,, %, BMSCs 5 μl Ligation Mighty Mix, 6, 30min, E.coliDH5α,.3.6 BMSCs Hind Ⅲ XhoⅠ, BMSCs2 0.25%,.3.4 pcdna3.-tgf-β pcd- NA3.-bFGF min - 5 min,, PBS 3 pmd20-nd2 pcdna3. PBS, BMSCs Hind Ⅲ XhoⅠ, ml -, FITC-CD295μL T4, FITC-CD905μL PE-CD345μL PE-CD455μL, :T 6 E.,4 5% CO 2 colidh5α,, 30min,PBS, ( ) 37 5%CO 2 5,,.3.7 BMSCs 37, μl PCR.3.5 SD BMSCs SD, 75% ( )208 2, 58 (EDTA), cm - 80%~00%, 3 BMSCs, 3 BMSCs 3 BMSCs,PBS min 0% DMEM, 5mL,200r bfgf TGF-β 3 BMSCs, Lipofectamine2000 5min,,.3.8 RT-PCR BMSCs TGFβ bfgf, DMEM/F2, BMSCs24h, RNA 5mL DMEM/F2,, bfgf TGF-β (, ) Trizol BMSCsRNA, 50mL, 20 ml 80, 50mL, r min - RNA, PCR PCR 20min, 20 ml 0.5% TGF-β bfgf β-actin bfgf 5 -ATGGCAGCCGGGAGCATCACCACGC-3 468bp bfgf 5 -TTAGCTCTTAGCAGACATTGGCAGG-3 TGF-β 5 -CTACCGCTGCTGTGGCTACTGG-3 476bp TGF-β 5 -TACAGCTCCACGTGCTGCTCCA-3 β-actin 5 -CGGGAAATCGTGCGTGAC-3 442bp β-actin 5 -TGGAAGGTGGACAGCGAGG-3

:TGF-β bfgf 25.3.9 Westernblot BMSCs TGFβ bfgf BMSCs 70% ~80%, 2 0 7 BMSCs ml RAPI, 0.0 mol L - TBST 3,ECL, BCA, 2.

4 :TGF-β bfgf Westernblot BMSCs TGFβ bfgf BMSCs 70% ~80%, BMSCs ml RAPI, 0.0 mol L - TBST 3,ECL, BCA, 2. BMSCs -70, BMSCs, 50μg, 2%Tris- SDS 24h, BMSCs, PVDF BMSCs 3d, BMSCs, 5% h,, ( TGF-β ( 2000) bfgf( 000), A) 5d, 4 0.0mol L - BMSCs( B) 6~7d,BMSCs TBST 3, HRP,, ( C) ( 5000),37 5%CO 2 h,, ( D) A:BMSCs 3d;B:BMSCs 5d;C:BMSCs 7d;D:BMSCs 2d 2.2 BMSCs BMSCs 98.2% 98.23%,, 3 CD34 CD % 0.0%, BMSCs CD90 CD CD29+CD34

5 南昌大学学报医学版２０１８年２月第５８卷第１期２６图３ＣＤ４５ＣＤ９０图４ＦＩＴＣ同型ＰＥ同型２３ｂｆｇｆＴＧＦ β１基因的扩增通过琼脂糖凝胶电泳进行检测结果显示４７８ｂｐ的条带和１１７３ｂｐ的条带与目的基因在大小上大致相同见图５

:TGF-β bfgf 27 ;8:pcDNA3.(+)-bFGF+pcDNA3.(+)-TGF-β M:DM2000bpmarker; 2:bFGF TGF-β, β -actin ;9:pcDNA3.(+), β -actin ;0:, β -actin ;:pcdna3.(+)-bfgf,tgf-β 5 bfgf TGF-β ;2:pcDNA3.

(+)-bfgf,bfgf ;2:pcDNA3.(+)-TGF-β bfgf, bfgf ;3:pcDNA3.(+)-bFGF+pcDNA3.(+ )- TGF-β,bFGF ;4:pcDNA3.(+),bFGF ;5:,bFGF ;6:pcDNA3.(+ )-bfgf, β-actin ;7:pcDNA3.(+)-TGF-β, β -actin pcdna3.

(+)-tgf-β pcdna3.(+ )-bfgf+pcdna3.(+ )-TGF-β 25ku, 8 M:DM 5 000(5 000 0 000 7500 5000 2500 000 250bp);M2:DM2000(2000 000 750 500 250 00bp);:HindⅢ pcdna3.(+) ;2:HindⅢ XhoⅠ pcdna3.

6 :TGF-β bfgf 27 ;8:pcDNA3.(+)-bFGF+pcDNA3.(+)-TGF-β M:DM2000bpmarker; 2:bFGF TGF-β, β -actin ;9:pcDNA3.(+), β -actin ;0:, β -actin ;:pcdna3.(+)-bfgf,tgf-β 5 bfgf TGF-β ;2:pcDNA3.(+ )-TGF-β,TGF-β ;3: 2.4 pcdna3.(+)-tgf-β pcdna3.(+)-bf- GF pcdna3.(+)-tgf-β pcd- NA3.(+)-bFGF HindⅢ XhoⅠ, 3, bp, M:DM 2000( bp);:pcDNA3. (+)-bfgf,bfgf ;2:pcDNA3.(+)-TGF-β bfgf, bfgf ;3:pcDNA3.(+)-bFGF+pcDNA3.(+ )- TGF-β,bFGF ;4:pcDNA3.(+),bFGF ;5:,bFGF ;6:pcDNA3.(+ )-bfgf, β-actin ;7:pcDNA3.(+)-TGF-β, β -actin pcdna3.(+)-bfgf+pcdna3.(+)-tgf-β,tgf-β ;4:pcDNA3.(+ ),TGF-β ;5:, TGF-β 7 bfgf TGF-β BMSCs 2.6 bfgf TGF-β BMSCs TGF-β bfgf pcdna3.(+)-bfgf pcdna3.(+)-bfgf+ 6 pcdna3.(+)-tgf-β 8ku, pcdna3.(+)-tgf-β pcdna3.(+ )-bfgf+pcdna3.(+ )-TGF-β 25ku, 8 M:DM 5 000( bp);M2:DM2000( bp);:HindⅢ pcdna3.(+) ;2:HindⅢ XhoⅠ pcdna3.(+)- bfgf ;3:HindⅢ pcdna3.(+) ;4:HindⅢ XhoⅠ pcdna3.(+)-tgf-β 6 :pcdna3.(+ )-bfgf+pcdna3.(+ )-TGF-β ;2:pcD- NA3.(+)-bFGF;3:pcDNA3.(+)-TGF-β ;4:pcDNA3.(+); 5: 8 bfgf TGF-β BMSCs 2.5 bfgf TGF-β BMSCs RT-PCR,bFGF 3 TGF-β SD BMSCs 7 [9],

7 28 [-2],,, BMSCs,, BMSCs [3],BMSCs, bfgf FGFs,,,bFGF MSCs, MSCs [7] Liu [3],,MSCs bfgf Nakahara [4] bfgf,,, Lee [5], bfgf BMSCs,, bfgf 48(7): TGF-β,,TGF-β ( ),, TGF-β [6] DNA, Park [7],TGF-β MSCs Ⅱ T, IL-6 TNF-α IL-7, TGF-β [8], MSCs [6] TGF β bfgf,,,,, TGF- β bfgf BMSCs, [] BotelhoJ,CavacasM A,MachadoV,etal.Dentalstemcels: [0], BMSCs TGF-β bf-, GF, TGF-β bfgf :,, recentprogressesintissueengineeringandregenerativemedi- cine[j].ann Med,207,49(8): [2] HuL,Liu Y,WangS.Stemcel-basedtoothandperiodontal regeneration[j].oraldis,207.[epubaheadofprint]. [3] ClarkeLE,McConnelJC,SherratMJ,etal.Growthdifer- entiationfactor6andtransforminggrowthfactor-betadiferen- tialymediatemesenchymalstemceldiferentiation,composi- tion,andmicromechanicalpropertiesofnucleuspulposuscon- structs[j].arthritisresther,204,6(2):r67. [4] ErcalP,PekozerG G,GumruOZ,etal.InfluenceofSTRO- selectiononosteogenicpotentialofhumantoothgermderived mesenchymalstemcels[j].archoralbiol,207,82: forminggrowthfactor-β signalingby mediatingrecyclingof thetypeⅠreceptor ALK5[J].PLoS One,207,2(): e talbonedefects[j].jorthopsci,205,20(4): [7] QianJ,Jiayuan W,WenkaiJ,etal.Basicfibroblasticgrowth factorafectstheosteogenicdiferentiationofdentalpulpstem celsinatreatment-dependentmanner[j].intendodj,205, [5] YangC,WangY,Xu H.Fluorideregulateosteoblastictrans- [6] YinC,ChenJ,ChenZ,etal.hBMP-2andhTGF-β expressedin implantedbmscssynergisticalypromotetherepairingofsegmen- [8] Du M,ZhuT,DuanX,etal.Acelulardermalmatrixloadingwith bfgfachievessimilaraccelerationofboneregenerationtobmp-2 viadiferentialefectsonrecruitment,proliferationandsus- tainedosteodiferentiationofmesenchymalstemcels[j].ma- tersciengc MaterBiolAppl,207,70(Pt): [9] VanDykeTE.Pro-resolvingmediatorsintheregulationofperio- dontaldisease[j].molaspectsmed,207,58:2-36. [0] MeyleJ,ChappleI.Molecularaspectsofthepathogenesisof periodontitis[j].periodontol,205,69():7-7. [] TakewakiM,Kajiya M,TakedaK,etal.MSC/ECM celular complexesinduceperiodontaltissueregeneration[j].jdent Res,207,96(9): [2] ChalisserryEP,NamSY,ParkS H,etal.Therapeutic potentialofdentalstem cels[j].j Tissue Eng,207,8: [3] LiuSB,HuPZ,HouY,etal.Recombinanthumanbonemor- phogeneticprotein-2promotestheproliferationofmesenchy- malstemcelsinvivoandinvitro[j].chin MedJ(Engl), 2009,22(7): ( )208 2, 58 ( 3 )

8 : 3 (SOD),,, :,, SOD [6] MDA [] LiJ,ZhongL,WangF,etal.DissectingtheroleofAMP-acti- vatedproteinkinaseinhumandiseases[j].actapharmsinb,, 207,7(3): , [2] Totzeck M,KorsteS,MinalainenI,etal.S-nitrosationofcal- [7] painsisassociatedwithcardioprotectionin myocardiali/rin- jury[j].nitricoxide,207,67:68-74., [3],. [J].,,205,37(8): [4],,,., [J].,, 203,5(8): [8], [5] YangZ,DuanZ,YuT,etal.Inhibitingcytochromecoxidase leadstoaleviatedischemiareperfusioninjury[j].koreancirc, J,207,47(2): [6] Totzeck M,HendgencotaU,RassafT,etal.Cardioprotection, byremoteischemicconditioning:mechanismsandclinicalevi- dences[j].worldjcardiol,205,7(0): SF SOD I/R,MDA [7] DongL Y,LiS,ZhenY L,etal.Cardioprotectionofvitexinon I/R, myocardialischemia/reperfusioninjuryinratviaregulatingin- flammatorycytokinesand MAPK pathway[j].am J Chin Med,203,4(6): [8],,,. [J].,,2003,20(2):43-45., ( : ), 櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐櫐 ( 28 ) [4] NakaharaT,NakamuraT,KobayashiE,etal.Novelapproach toregenerationofperiodontaltissuesbasedoninsitutissue engineering:efectsofcontroledrelease ofbasicfibroblast growthfactorfrom asandwich membrane[j].tissue Eng, 2003,9(): [5] LeeJH,LeeYJ,Cho HJ,etal.TheincorporationofbFGF mediatedbyheparinintopcl/gelatincompositefibermeshes forguidedboneregeneration[j].drugdelivtranslres,205, 5(2): [6] CraneJL,CaoX.Bonemarrow mesenchymalstemcelsand TGF-βsignalinginboneremodeling[J].JClinInvest,204, 24(2): [7] Park MJ,Park H S,Cho M L,etal.Transforminggrowthfac- torβ-transduced mesenchymalstem celsameliorateexperi- mentalautoimmunearthritisthroughreciprocalregulationof Treg/Th7celsandosteoclastogenesis[J].ArthritisRheum, 20,63(6): [8] KhaghaniS A,Denyer MCT,YousefiM.Efectoftransfor- minggrowthfactor-β inbiologicalregulationofprimarychon- drocyte[j].amjbiomedeng,202,2():-8. ( : )

4 : pcdna3.1(+) ;PLV-EF1A-EGFP(2A) 30s,41 30s,72 2min,35 ; Puro ; TG1; (Hu min 1% manembryonickidney293cels); (Ma- din-darbycaninekidneyce

4 : pcdna3.1(+) ;PLV-EF1A-EGFP(2A) 30s,41 30s,72 2min,35 ; Puro ; TG1; (Hu min 1% manembryonickidney293cels); (Ma- din-darbycaninekidneyce 33 4 2017 6 CHINESEJOURNALOF VIROLOGY Vol.33 No.4 June 2017 1# 2#,, 2, 2, 1 3* 1*,, (1., 511436; 2., 510230; 3., 510663) : HEK-293-HA RT-PCR HA, pcdna3.1(+ ), pcdna3.1(+ )-HA pcdna3.1(+)-ha PLV-EF1a-EGFP(2A)Puro