f zu{ g } 15 í } 23 2011 06 04 Çf Journl of Clinicl Rehilittive Tissue Engineering Reserch June 4, 2011 Vol.15, No.23 ƒ Œ Œý Œq ýéä* Ê Ð 1 2 ¼ é 2 é 2 Ð 3 Directionl differentition of dipose tissue-derived stem cells into heptic-like cells in vitro y procedure-induction Liu Jin 1, Li Li 2, Rn Jing-hu 2, Zhng Sheng-ning 2, Sho Jin-chun 3 Astrct BACKGROUND: How to estlish effective stle heptic cell differentition schedule, to purify or rpidly mplify stle heptic-like cells needs to e solved efore dipose tissue-derived stem cells (ADSCs) were used to tret liver diseses. OBJECTIVE: To estlish sequencing induction system of ADSCs trnsforming into heptic-like cells. METHODS: After isolted nd purified the Lewis rt ADSCs, the surfce mrks of ADSCs were identified y flow cytometry. The rt ADSCs were trnsformed into the heptic-like cells in the procedure-culture system y tris-step including heptocyte growth fctor (FGF), firolst growth fctor-4, cid firolst growth fctor nd oncosttin M cytokine. RESULTS AND CONCLUSION: After rt ADSCs were induced t 7, 14 nd 21 dys, the expression of lumin (ALB), lph fetoprotein (AFP) nd CK18 mrna ws determined nd fortified over time. Heptic-like cells hd ALB synthesis function. Metolism of mmoni nd ure synthesis occurred nd lsted fter 912 dys. Results suggest ADSCs were successfully differentited into heptic-like cells in vitro fter procedure-induction. Liu J, Li L, Rn JH, Zhng SN, Sho JC. Directionl differentition of dipose tissue-derived stem cells into heptic-like cells in vitro y procedure-induction.zhongguo Zuzhi Gongcheng Ynjiu yu Linchung Kngfu. 2011;15(23): 4237-4241. [http://www.crter.cn http://en.zglckf.com] nœ Œ o ŒooiÐ z q ŒÉä ä ³ Œz q Œ}ÁÈq À rq ÙŒ Œ±äg Œqz ä ƒ Éy ä Lewis ÙŒ Œ Œy¹ ºŸÅ É 3 fâ Ø ÿ ŒmÀ ŒmÀ 4 ŒmÀ Íp M Œ q»ƒ ˆŒ Œý Œ±ä ÙŒ Œ 7 14 21 d ý ŒÂ Ÿ² ALB AFP CK18mRNA Ÿ² à  À Œº q qü ØŒ x qü ØŒ 9~12 d Çj Ÿ Œ Œ ƒ É ù رäg Œ ºÀ Œ Œ Éä Œ z ä Ù doi:10.3969/j.issn.1673-8225.2011.23.014 ÊÐ ¼ é é Ð.ƒ Œ Œý Œq ýéä[j].f zu{ g 2011 15(23):4237-4241. [http://www.crter.org http://cn.zglckf.com] 1 Kunming Medicl 650031, Yunnn Province, Chin; 2 Deprtment of Heptopncretoiliry Surgery, Gnmei Hospitl Affilited to Kunming Medicl Province, Chin; 3 Deprtment of Clinicl Lortory, Gnmei Hospitl, Kunming Province, Chin Liu Jin, Studying for doctorte, Attending physicin, Kunming Medicl College, Kunming 650031, Yunnn Province, Chin liujinwxc1219@163.c om Correspondence to: Li Li, Professor, Chief physicin, Deprtment of Heptopncretoiliry Surgery, Gnmei Hospitl Affilited to Kunming Medicl Province, Chin ynkmlili@yhoo.com 0 Œ Œž ŒöÎÎ Œ qu{f öjâ«œ ý Œ±äq Œ [1] Œ Œ(dipose tissuederived stem cells ADSCs) ² v q yâ«œ f º Äö i ~pîîâ«œ d k ÂÍ h ³ADSCsŒ Éäg Œ [2] ü ik ² u{ 1 Œ ƒ Í Âö p200901/201003 ç àmˆçã Íy Œ» Í Ï y Ïöy Œñ Willm's E» Ù Ù CD44-FITC ƒ Ù Ù CD34-FITC ƒ Ù Ù CD29-FITC ƒ Ù Ù CD49d-FITC ƒ Ù ŒmÀ ŒmÀ Íp M ö± Ïr Ùq q FITC q ˆ IgG ƒ Œy ˆ À ˆ Sigm ¹ú ˆ GIBCO ¹ú ˆ Millipore ¹ú ˆ Biolegend ¹ú ˆ RD ¹ú ˆ Promeg ¹ú ˆ Acm ¹ú ˆ Millipore ¹ú ˆ BEKMAN Fc-500 OLYMPUS ¹ú Supported y: the Scientific Reserch Foundtion of Deprtment of Science nd Technology of Yunnn Province, No. 2007CA007* Received: 2011-02-26 Accepted: 2011-05-09 ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 4237
1 ä ä 650031 2 ä œ } Þ ä 650011 3 ä Æ Þ ä 650011 öè 1973 ä ä x Þ } Ôd liujinwxc1219@ 163.com ìí Ì ä œ } Þ ä 650011 ynkmlili@yhoo. com f É ú:r394.2 h u:b ú:1673-8225 (2011)23-04237-05 2011-02-26 º 2011-05-09 (20110226008/MeQ) 4238 ÍÙg Ÿ 4 ÚÄ Lewis Ù 10ù näs ³Ìé ÍÙg¹ú Ͳzf Ùg ˆõd y µ 2006 ö q ºp ÍÙgq ÙADSCsÉy ä ñx»ö¹ h[3] 4 ÚÄ Lewis Ù çq Œ PBSÀ ýó 1 mm 3 NH 4 Cl à Œ Œñ ä1 h Ø üƒz u» (ƒ DMEM ÿƒzé g10% fž ) 200 μm}ˆ² y Œ Éy Œn Âz ö ž» Ã ä Œ Ž 70%~80% ~x }3~6xADSCsÍ ê Œ Œ g10 9 L 1 Ø Ù ÙCD44 CD29 CD49d CD34-FITC ƒü10 μl 4 30 min Œy ŒŸÅ ÙADSCsý Œq }3x ADSCs x10 4 / yp6 ŒÀ Ž70%~80% z ä }  ž» ¹»48 h Œ Ð ü ä }pâ Willm's E»»(pH 7.2)e»ƒ fÿ50 mg/l Ù ŒmÀ 10 mg/l ŒmÀ 1 10 mg/l ŒmÀ 4 Œ ±¾ qv¾(its) 3 d 1 }  ²»ƒ Ø 10 mg/l Íp 10 mg/l Ø 2.5 ml 3 d 1 21 d ä ØÿƒzÉ g5% m fž qwillm's E»» ŒÀ Ž70%~80% ~x ~Ž}3xý Œ ²Ÿ o ä Œ ²zfqÙ ¹ ˆ À Œ RT-PCR 0 14 21 28 dý Œ RNAŸRT-PCR ALB AFP CK18 mrnaÿ² xgadphgõ nprimer 5.0 g dpromeg¹úö± Ïr 30 10 min 42 9 min 99 5 min 4 5 min ö ¼x cdnag ŸPCR 94 2 min 94 60 s 55.8 60 s 72 60 s x 4 Î 35f j 72 10 min 1%kŒ ÄŒn g Ê g g ALB AFP CK18 GADPH f öž 6 f 1 2 3 Ø ýq Œ g Í 4 5 6 Ø qadscs g d ü»18f»»}3 6 9 12 15 18 21 24 27 9f Í ö d ü²6f Í» Ø 5 mmol/l NH 4 Cl 24 h ¹Ž ÙämäÉ y öž f e ²Ÿ É o ä 6 f 1 2 3 g Í Ø ýq Œ 4 5 6 Ø qadscs g d ý28 d d ü f Ç Œff 40 g/l Šn 30 min 0.1% Triton-X100 u ž Á15 min Ø Ùq q ƒ ˆ 4 À~f² 37 30 min PBSÀ 3 Ø FITC q ˆ IgG ƒ 37 30 min Àm DAPI Œ À  Œ É f q nx _ ±sÿx f q ²Ÿ  ± nanovaé P < 0.05g i P < 0.01g Å i nspss 13.0 ±z k 2 CTT CAA AGC CTG GGC AGT AG TGG AGA AGT GCT CCC AGT CT ATT GCC AGT TCT GGG TTG AC GGT GAA GGT CGG AGTCAACG GCA CTG GCT TAT CAC AGC AA GCA GTG GTT GAT ACC GGA GT TTG AGT TGC TCC ATC TGC AC CAA AGT TGT CAT GGA TGG ACC w À (p) 221 359 335 450 2.1 ADSCs ëc Ç Ù ADSCsÉy Ã}²ý1 µé96 Ç j Œq Çjig5.13%~9.47% Œ g Ÿg Ê Œ 1 14~21 dë ~x À ~x(10xx ) P.O. Box 1200, Shenyng 110004 cn.zglckf.com
ŒŸjÇmÀ r 2 Œ Âg (36.67±3.27) h Œ Éy»qADSCs 3~6 x Œ Î Ÿ²CD29(98.92±0.63)% CD44(99.23±0.63)% CD49d(99.23±0.37)% CD34 ƒ Ÿ²(0.96±0.87)% Figure 4 At 2 wk fter differentition of rt dipose tissuederived stem cells into heptic-like cells ( 200) 4 ÙŒ Œý Œ 2 ( 200) Figure 1 Rt dipose tissue-derived stem cells clone 1 wk fter isoltion ( 200) 1 Éyq ÙŒ Œ» 1 qâ à ( 200) : 200 : 400 Figure 5 At 3 wk fter differentition of rt dipose tissue-derived stem cells into heptic-like cells 5 ÙŒ Œý Œ 3 Figure 2 The tenth pssge of rt dipose tissue-derived stem cells fter isoltion nd purifiction ( 200) 2 Éy äq Ù} 10 xœ Œ( 200) 2.3 RT-PCR ÆgΫ14 d Çjñ Œ AFP ö d Œ Al CK18 Ÿ ² AFP ALBöCK18Ÿ²Ã q  À Ÿ²² 6~8 2.2 ADSCs Ç 1 ý Œ Œ«Çq «Ø Œ² n ŒmÀ ² ü µé Œ Œ«3 221 p Figure 6 Alumin mrna PCR electrophoretogrm 6 ALB q PCR n Figure 3 At 1 wk fter differentition of rt dipose tissuederived stem cells into heptic-like cells ( 200) 3 ÙŒ Œý Œ 1 ( 200) 7~14 dçj Œ Œmfu ŒˆmÀ 4 e ý ŒÄ màz É Œ Œ /Œ«Ã mž Œ } }3 }pâ ý ŒmÀØ d Œ /Œ«²  ù Œ ²n Ž21 dý Çj Œ 5 5~7 dý~x 359 p Figure 7 Alph fetoprotein mrna PCR electrophoretogrm 7 AFP q PCR n ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 4239
335 p öj Œ«g â Ÿ ýq ŒŒÉ Œg q ADSCs ƒ o Œ 11 Figure 8 CK18 mrna PCR electrophoretogrm 8 CK18 q PCR n 2.4 À Š» f 12 dxý d ±  i (P < 0.05) iýã Âq À 9 9 dý Çj ü 12 dý d ± é(p < 0.05) à  À ºrm³i² é(p < 0.01) 20 dýçjî ±Î 10 2.5 ê ALB ΫALB o 4240 Concentrtion of mmoni (mmol/l) 7 6 5 4 3 2 1 0 Experimentl group Control group 3 6 9 12 15 18 21 24 28 30 Time (d) P < 0.05, P < 0.01, vs. control group Figure 9 Concentrtion of mmoni in superntnt fluid of the experimentl nd control groups 9 Í d f q ä Concentrtion of ure (mmol/l) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Experimentl group Control group 3 6 9 12 15 18 21 24 28 30 Time (d) P < 0.05, P < 0.01, vs. control group Figure 10 Concentrtion of ure in superntnt fluid of the experimentl nd control groups 10 Í d f q ä Figure 11 Expression of lumin y fluorescence immunohistochemisty in the heptic-like cells( 400) 11 Œ ALB o ( 400) 3 Seo} [4] p2005 Ë ÇoADSCsý Œ ýéäqùÿ ý ¼ n ü ˆ ADSCsý Œ ýéä ü i ö ŒqmÀ À ~x»ý Œ ØŒ ä [5-6] g Égƒ¼ ƒ ey² ƒ¼ šù Í j ³² ŒÂ س Œý ŒÉä ŒýÅ i ƒ p} ŒiÂq Œ ± q Ä Äu{ Œg ƒ ù v Í ù Œ ƒ Œg [7] Œqƒ ²zèÉ Ä Í Œ ŒØŒŸ²iÂq Ÿ Ìp ŒÐƒ ŒmÀqƒ ²ü ŒØŒqŸ² öir q Œ ömà Œý ŒÉäx ö ŒqmÀµ q [8] ºf ŒmÀ Œ ²zf n q ºg ŒKupffer Œ ¼q Œfrm Œ q dé Ï ŒmÀ µ«ŒÉäqu º ƒc-metö ERK1/2 P38 P13k/Akt À ³² ² Œ² G 1 S np38 ÍÏSB203580ù Í Œ [9] ŒmÀ t Œq¹ŒÉä g q ºÀ i ŒmÀ º ƒù³² PI3K/Akt/PKB ú³ Î Œ «É Ÿ², t Œqq¹ŒÉ äg [10] ŒmÀ Œ Å g q ŒÉä ŒmÀ Œq Éä  Œýºw ýéä e éü²nù x Œý Œ ýéä Íp MŒ ŒÉägº üyx ØŒq d Œ Œ [11] yü qq«œ ³² Í Œ P.O. Box 1200, Shenyng 110004 cn.zglckf.com
É ²Éä ŒŸ² ŒqŸ ƒ q É ² ƒ Ä Œq y Ø q Â Ï ö q² ü Í ¹Ðtq Í u Øx~ä ² zf noz äéâ x Œý Œ ýéä ü ŒqÉä Œ  ²ä ý Œ 1 ý ²n ² Œ d q Œ }  ³² Þ q k ˆ Œ Éäg ˆ Œü ä } pâ ³² ŒmÀ ö ŒmÀ q éü n ˆ Œý Œ ýéä } Â Ê Ã ŒmÀ ü Ø Íp Mö Ø ýq Œ ö d [12-13] Í öj ADSCs³² v Œ² ö q Œ Œy ýv Œ rqmàœ 4~7 d~x1 10x RT-PCR x 14 dýalb AFP CK18  Ÿ² Ãs  À Œy ƒ Œ ~xý o x ŒALB Œ fqÿ² gâ Éä q Œ uº ü ØŒ ü º x ØŒ ØŒŸ ²qg g«œ ý ²Ÿ yx ±ä ü Œ Í o ýq Œ Œº q ±ä qœ Œ ü ƒ ŒqmÀ ŒöÉäŒ Å z q à ² y k q» ÿ v qä É ÿž ˆnü qž x g Ø q Œ Œ mà [8] Í ƒ ²no²ü ŒmÀqWillmE» Ø ž  uˆn±ƒ ž ü ä få qâ ŒmÀŽ60%~ 70% ²Ÿ ž»ü ä À²21 dq ² zf Œ ² mà³ ² ~ Ž Œ Í öm Œ²ä ù q h Ç ADSCsq ²zfùx»m ˆ Œ Ø«Íf ±oey öj Ä n röœ Œ ²äŸ Œ ³²«}²ö ÃmÀö ùx ä q Œ ² j èé ² e ÍpH ö ä Âk Å q xadscsý Œ qºƒ ² väö Œ Ø 4 õ h [1] Ochiy T, Ymmoto Y, Bns A.Commitment of stem cells into functionl heptocytes.differentition. 2010;79(2):65-73. [2] Lopez MJ, Spencer ND.In vitro dult rt dipose tissue-derived stroml cell isoltion nd differentition.methods Mol Biol. 2011; 702:37-46. [3] Zhu Y, Liu T, Song K,et l. Adipose-derived stem cell: etter stem cell thn BMSC.Cell Biochem Funct. 2008;26(6):664-675. [4] Seo MJ, Suh SY, Be YC,et l.differentition of humn dipose stroml cells into heptic linege in vitro nd in vivo.biochem Biophys Res Commun. 2005;328(1):258-264. [5] Bns A, Tertni T, Ymmoto Y,et l. Rpid heptic fte specifiction of dipose-derived stem cells nd their therpeutic potentil for liver filure.j Gstroenterol Heptol. 2009;24(1): 70-77. [6] Bonor-Centelles A, Cstell JV, Gómez-Lechón MJ.Adipose tissue-derived stem cells: heptic plsticity.gstroenterol Heptol. 2008;31(5):299-309. [7] Ruiz JC, Ludlow JW, Sherwood S,et l. Differentited humn dipose-derived stem cells exhiit heptogenic cpility in vitro nd in vivo.j Cell Physiol. 2010;225(2):429-436. [8] Dong XJ, Zhng H, Pn RL,et l. Identifiction of cytokines involved in heptic differentition of mbm-mscs under liver-injury conditions.world J Gstroenterol. 2010;16(26):3267-3278. [9] Peur M, Bizik J, Slmenperä P,et l. Bone mrrow mesenchyml stem cells undergo nemosis nd induce kertinocyte wound heling utilizing the HGF/c-Met/PI3K pthwy.wound Repir Regen. 2009;17(4):569-577. [10] Zhou QJ, Hung YD, Xing LX,et l.in vitro differentition of emryonic stem cells into heptocytes induced y firolst growth fctors nd one morphologicl protein-4.int J Biochem Cell Biol. 2007;39(9):1714-1721. [11] Song HY, Jeon ES, Kim JI,et l. Oncosttin M promotes osteogenesis nd suppresses dipogenic differentition of humn dipose tissue-derived mesenchyml stem cells.j Cell Biochem. 2007;101(5):1238-1251. [12] Bonor-Centelles A, Jover R, Miret V,et l. Sequentil heptogenic trnsdifferentition of dipose tissue-derived stem cells: relevnce of different extrcellulr signling molecules, trnscription fctors involved, nd expression of new key mrker genes.cell Trnsplnt. 2009;18(12):1319-1340. [13] Chivu M, Dim SO, Stncu CI,et l.in vitro heptic differentition of humn one mrrow mesenchyml stem cells under differentil exposure to liver-specific fctors.trnsl Res. 2009;154(3): 122-132. Ž Èq «Ù ÞÁ Þ ¾Ð (2007CA007) qœ î { ÇÁ Ô rd «h  c c c ò À d n d Ž ä œ Æ Þ Á d ÌrÀ{ ß À jãà ½ Ò ͽÅÍ Ò½í d k  «n t üì d Ë q c c c r Æ w Æ pumed á 200201/201101 Î «57 í Ô ƒ q c c c r Æ w Ϋ8 «Ô ~ ÔÌ q c cn À r Æ w Ϋ0 œ u d u ƒ { óu Ä { óu Ó É t ÍŠ ÏÇò n n nòê «ò À Ç d u d ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 4241