2 ( )208 58 JournalofNanchangUniversity(MedicalSciences)208,Vol.58No. TGF-β bfgf,, 2 (., 330006; 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 : 2095-4727 (208)0-0022-07 DOI:0.3764/j.cnki.ncdm.208.0.006 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. :207-09-3 : (200BSA5900) : (983 ),,,, :,,E-mail:song36079969@26.com
: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 030684.,NC 000004.2), Primer5.0 HindⅢ XhoⅠ ( ) TGF-β :F 5 - RNA Trizol,,
24-80, PBS,20 400r min - 0 min, RNA, PCR,, 2 0 5 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, 5 0 5 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), 2 0 3 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,20 800 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. 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 CD45 0.00% 0.0%, BMSCs CD90 CD29 2 4 2 CD29+CD34
南昌大学学报 医学版 ２０１８ 年 ２ 月 第 ５８ 卷第 １ 期 ２６ 图 ３ ＣＤ４５ ＣＤ９０ 图 ４ ＦＩＴＣ 同型 ＰＥ 同型 ２ ３ ｂｆｇｆ ＴＧＦ β１ 基因的扩增 通过琼脂糖凝胶电泳进行检测 结果显示 ４ ７ ８ｂｐ 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.(+ )-TGF-β,TGF-β ;3: 2.4 pcdna3.(+)-tgf-β pcdna3.(+)-bf- GF pcdna3.(+)-tgf-β pcd- NA3.(+)-bFGF HindⅢ XhoⅠ, 3, 5427 478 73bp, M:DM 2000(2000 000 750 500 250 00bp);: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(5 000 0 000 7500 5000 2500 000 250bp);M2:DM2000(2000 000 750 500 250 00bp);: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],
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