f zu{ g } 15 í } 14 2011 04 02 Çf Journal of Clinical Rehabilitative Tissue Engineering Research April 2, 2011 Vol.15, No.14 g ²Îε«Œý ŒqÉä* 1 é 1 2 fi 2 z 2 Ä 2 1 2 A natural activity factor promotes cardiogenic differentiation of bone marrow derived mesenchymal stem cells Peng Peng 1, Mo Ye-hua 1, Lin Xiao-li 2, Li Zhong-yu 2, Cheng Lei 2, Zhou Zhen 2, Li Ming 1,2 Abstract BACKGROUND: Previous studies have demonstrated that the methanol extract of Geum japonicum (EGJ) can effectively promote the cardiogenic differentiation of the exogenous mesenchymal stem cells (MSCs), leading to substantial repair of infarcted heart with cardiac function significantly improved. In this study, the isolation of the active compound, which can induce cardiogenic differentiation of MSCs in cell culture system, was described through a bioassay guided fractionation strategy. OBJECTIVE: To isolate active compound (cardiogenin) from Geum japonicum and to observe its activity in promoting cardiogenic differentiation of MSCs. METHODS: The MSCs were isolated from the bone marrow of SD rats. The isolated MSCs were cultured and treated with EGJ (100 mg/l) and cardiogenin (10 mg/l) respectively. Immunofluorescent staining with antibodies specific for early cardiac differentiation marker (MEF2) and myosin heavy chain (MHC) was performed at 3 and 7 days after treatment respectively to monitor the process of cardiogenic differentiation of MSCs. RESULTS AND CONCLUSION: It was found that approximately 39% of both EGJ and cardiogenin treated MSCs were positively stained with MEF2 at 3 days after treatment. On day seven of the treatment, more than 70% of both EGJ and cardiogenin treated MSCs were positively stained with MHC specific antibodies. The cardiogenin can significantly induce cardiogenic differentiation of MSCs in vitro as does EGJ, but the dose of cardiogenin required for induction of cardiopgenic differentiation of MSCs is much less but the induction efficiency is little higher than that of EGJ. Peng P, Mo YH, Lin XL, Li ZY, Cheng L, Zhou Z, Li M. A natural activity factor promotes cardiogenic differentiation of bone marrow derived mesenchymal stem cells.zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(14): 2535-2539. [http://www.crter.cn http://en.zglckf.com] x u{ q Ùƒ¼ g EGJ Œ ² qîî⵫œéä Œ È ² Éy EGJ Í g q cardiogenin º ÿkœ ²Âµ«Œý ŒÉ ä rq Éy q cardiogenin º ²Îε«ŒÉä Œq n Éy ä ÙqÎε«Œ ÉÌn q g EGJ 100 mg/l cardiogenin 10 mg/l k µ«Œ k⵫œq} 3 } 7 n o q ²Ÿ Éäg g o Ÿ kq⵫œ ÿÿ² g g k 3 d ý EGJ cardiogenin 39%q ŒŸ² Éä qg g Mef2 } 7 EGJ cardiogenin 70%x q ŒŸ² Œq k q MHC x EGJ cardiogenin Œ ÎΠµ«œý ŒÉä cardiogenin qn EGJ 5~10 Éäq iknî ºÀ g ž Œo Îε«Œ Œ cardiogenin doi:10.3969/j.issn.1673-8225.2011.14.015 é fi z Ä. g ²Îε«Œý ŒqÉä[J].f zu{ g 2011 15(14):2535-2539. [http://www.crter.org http://cn.zglckf.com] 1 Key Laboratory for Province, China; 2 Laboratory of Innovative Medicine, Hong Kong Special Administrative Region, China Peng Peng, Studying for master s degree, Key Laboratory for Province, China xuanxuan86120@ 163.com Correspondence to: Li Ming, Doctor, Professor, Doctoral supervisor, Key Laboratory for Province, China; Laboratory of Innovative Medicine, Hong Kong Special Administrative Region, China li.ming@labinnovativ emedicine.com 0 ý Œ Ãr³ r à Ž ،ƒ ö ˆ Ÿ öm Œüx Œ rÿ g Éä Œ ý Œ³² Œ¼m Œ po q Ãs ŒØŒq² ƒ ç ŒØŒ² ä ~ u{ x ý q Œ d É [1-3] xö m³ xrm iq Œz šœ x ƒz Î d«n «} Ê Äx g ³² Œ z Ø ØŒq Œ r g ýq o o rðùœnp Œz oq Œ Œ Œ ÎÎ Œ} [4-9] ŽÎ Îqµ«Œnp o ö vžƒ Ä ƒ k ± ùx ¹ n Œz ÅgqkÁ È Žpqíà o µ«Supported by: the RGC grant (2140569) from Hong Kong Government Research Committee No. 470707* Received: 2010-12-14 Accepted: 2011-01-28 ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 2535
www.crter.org }. g ²Îε«Œý ŒqÉä 1 { Ì ƒ 510632 2 Ÿu Ÿ 1986 µ { Þ Á x ¾ Ôd xuanxuan86120 @163.com ì Ì { Ì ƒ Ÿ u Ÿ li.ming@labinnov ativemedicine. com f É ú:r394.2 h u:b ú:1673-8225 (2011)14-02535-05 2010-12-14 º 2011-01-28 (20101214011/MeZ) 2536 Œý Éäq iå ƒ e Í o µ«Œ o q n nz ŽƒÎε«Œq o Ùg š Œöjnz qîîâµ «ŒÉä Œ d²q q m Œ g É p ž q² ç xrm iq x ŒØŒq n x ùx ² µ«Œý Éä iq g Å È Ð qu{ gegjœ qîî⵫œéä ØŒq Œ [10] ³² Í qé y ² Éy ÍoEGJq cardiogenin º ÿœ Îε«Œ Éä Œ Í cardiogeninùx µ«Œý É ä ±ocardiogenin EGJ µ«Œý Éäq i Œq Ÿ d Í Âö Íp201002/11 d Îy Ë ç Í 3 ÚSPF SD Ù2ù Ä ƒ «200~250 g n Æ ÍÙgf Ͳzf Ùg ˆ}ü2006 y µö q ºp ÍÙgq [11] Í cardiogeninqéy 2009 p «Ä e Óv n 7 d n Ä Íq p f n { j j Ãï º 4 fëé n 4fËÉ Âµ«Œý ŒÉäq Í vf ²Ç qëé nsephadex LH-20 ²f qëé² Éy vƒzé g10% n Œ º 7f É n²7f É ²Ÿ µ«Œý ŒÉäq Í ² }² ý }² Çq É gcardiogenin nx ²qý Í ÙÎε«ŒqÉy ä 3 Ú Ù È Î ŒÎ nî µé ²³ Í qpbsýz  úf z n ž¾ Î e ÅÇÎÎ nÿƒzé g10% fž qimdm» À ÎÎŽ50 mly ~f ö À Å 2 000 r/min y 5 min ô ¼Ø 3 mlÿƒzé g 10% fž qimdm ±± Ø Ficollf 2 000 r/min y 30 min Ég3 g Œ fâ Ø grq Œ ÇŽy ~f n PBS e yp25 cm 2»mf ˆ37 ƒzé 95%{ ƒzé 5%CO 2 q» ~f» }2 }3 ÉÌ xý 3 d 1 ŒmÀŽ r 80% ² Ÿ~x» Ø ÿedtaq0.25%œ 37 ä1 miní Ç ± m Ø ÿƒzé g10% fž qimdmf ä 2 000 r/min y 5 min ~x3 4 ý Œ Ž g± q⵫œ ñ x» Œ gp0 }1 ~xq Œ gp1 x Ínqµ«Œ g P5Ðq Œ g k Œ P4 Œ yp µ r coverslipq24 f Ég100 mg/l EGJ» 3 d 7 d 10 mg/l cardiogenin»3 d 7 d 5%DMSO d k3 d 7 d EGJ cardiogenin p5%dmsof ¼Ø ÍÿƒzÉ g10% fž qimdm» f²ÿ Œ» 1 000 μl(ÿ Œ 1 10 5 f)» ˆp37 ƒzé 95% { ƒzé 5% CO 2 q»~f»12 h ý Œ kq}3 }7 ÉÌ Œ ²Ÿ g Œqn ÿ u ÿ Ê Œ } Œ o Œ»Ž}3 }7» nƒzé g4%qn 15 miný nèáqpbs 2 5 min/ n0.25%q Triton X-100 10 min ¼n PBS 3 5 min/ n1% BSA Á30 miný Ø 4 ² (Mef2 MHC g1 500z ) PBS 3 5 min/ p CY5 FITC g1 1 000z 1 h PBS 3 5 min/ ýn DAPI (1 100z ) 10 min npbs 1 ý n Ï À d P.O. Box 1200, Shenyang 110004 cn.zglckf.com
}. g ²Îε«Œý ŒqÉä www.crter.org 2.1 h ñx»q⵫œg ˆ À öj Ë yq⵫œ f Œ 24 hý µ É Œ m «iý Œƒz ÃmÀ jç Ê z g ý q ƒ»2 d 3 dq r ü غ 1a»5~8 d g Œ n 1b»9~14 d µ«Œù f»r ~xiýxü q mà 1c 7~9 dý²ÿ}1 ~x ~xý3 d íù ¹ 7 d EGJ 100 mg/l ( 3a) cardiogenin 10 mg/l ( 3b)ù 70%x q Œ À g À g Œ Œ Œ ý a: EGJ group b: Cardiogenin group Figure 3 Morphology of mesenchymal stem cells at 7 d after induction ( 40) 3 7 d e µ«Œ ( 40) d Œ z ƒ g ý qg d»²zf Œ Ø 4a b a: 3 d b: 6 d a: 3 d b: 7 d c: 11 d Figure 1 Morphology of mesenchymal stem cells at 3, 6 and 11 d after primary culture ( 40) 1 µ«Œñx»} 3 6 11 q ( 40) ý⵫œq g ý⵫œ mã ² pž 3 d úq EGJ 100 mg/l ( 2a) cardiogenin 10 mg/l ( 2b) ù 50%x q⵫œnñ Ê g ŒŒƒf a: EGJ group b: Cardiogenin group Figure 2 Morphology of mesenchymal stem cells at 3 d after induction ( 40) 2 3 d e µ«Œ ( 40) Figure 4 Morphology of mesenchymal stem cells at 3 and 7 d after induction in the control group ( 40) 4 3 d 7 d d µ«Œ ( 40) 2.2 Ϋn cardiogenin»âµ«œ3 dý 40%~50%q Œƒ f g g d ý Éäqg gmyocyte enhancer factor-2 (Mef2) Í} 7 70%~80%q Œƒ f g g d êƒqg g k q (MHC)Ÿ²Â r i d µ«Œ}3 }7 Mef2² MHC o gâ EGJ µ«Œ»3 dý 40%qµ «Œ g d}3 Mef2Ÿ²Â }7 MHCŸ²Â ù EGJ cardiogenin Œ µ«Œý ŒÉä cardiogeninqn EGJ 5~10 Éäq iknî 5 6 ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 2537
www.crter.org }. g ²Îε«Œý ŒqÉä 2538 a: DAPI staining, control group c: DAPI staining, cardiogenin group b: DAPI staining, EGJ group d: Mef2 staining, control group e: Mef2 staining, EGJ group f: Mef2 staining, cardiogenin group Figure 5 Immunofluorescent staining of mesenchymal stem cells at 3 d after induction ( 200) 5 µ«Œ 3 d ý Œ o ( 200) a: DAPI staining, control group c: DAPI staining, cardiogenin group b: DAPI staining, EGJ group d: Mef2 staining, control group e: Mef2 staining, EGJ group f: Mef2 staining, cardiogenin group Figure 6 Immunofluorescent staining of mesenchymal stem cells at 7 d after induction ( 200) 6 µ«Œ 7 d ý Œ o ( 200) ÍrÐg v { g o ùx o g o q g Å o o o ž~ Ì tg à Œ ØŒxöà m Ù ²Í Œ ž ØŒ ¹qrq Œ¼mÇ ØŒq Œ x q ² ~ «q o np Œ ¼mŒ ¼mŒ ƒ mžùx n ùœ n Í Œz g y q º ƒ qý Éäq i ˆº o Ío Œöj ²Âµ«Œý Éä iq g ùœ n⵫œz o qdn mè g o o Geum japonicum Thunb. var.chinense F.Bolleg y ²Å g f pê «} É ØŒgŸšr xéã p g oo q É o o oo [12] È qu{ÿ gegjœ ØŒ n o2è Âö Ì(PCT) ro180f [13-14] Íf Ìn ü Œ»rØ nq Ì ²ŸÉy»± q⵫œ ÎÎf Œ É ÎÎ Œg â ³ž Œ µ«Œ ºfµ«Œ f qº ý ÉäŒ q Œ q z ŒÉägž~ ¼q Œ Œ Œ Œ Œö Œ} [15] Í ŒŸÅq g²ÿ o wjé² Îqµ«Œ np⵫œ g qÿå g ˆ²y g ÍfÄx Í q n Íf Ficoll v q y ˆ Äq Œ ÍË qéy ä ² q äê n ³«Íµ«ŒÀ mý Øtrypsin EDTA»~¼ ä1 min  µ«œ Ÿ ä y ýyp m ö ~x äž}5x úù ± µ«Œ äý q⵫œ ±ê g z Œƒ± Œ«ƒ ùx g o fƒ» ä⵫œqz Í Ÿ cardiogenin k⵫œ3 d ý ù µ«Œ öm ä n zgý g± Œƒnñ q«oœƒ ÇÇ Œ«q Œ q g ŒmŽù Œ q Cardiogenin EGJ ²Âµ«Œý ŒÉä P.O. Box 1200, Shenyang 110004 cn.zglckf.com
}. g ²Îε«Œý ŒqÉä q ± Íf ÃoCardiogeninqn 5~10 p EGJqn x p ²Âµ«Œý Éäq Ÿj Žq d²qý Éäq ŒµŸ ² Œg q gmef2 MHC EGJf ÿ qù ²Âµ«Œý Éäq cardiogenin p Ão µ«Œý Éäq g Œq Žx d µ«Œý Éäq µ Žq g Œý ù Œ o Œ¼ Êq ²q q Ù s⵫œý ŒÉä déä i²ío 50%~70% ü k xo cardiogenin ùœ ƒ¼ ²¼ µ«Œý ŒÉä ² ² q Œ¼¼m mq Œ v q Œ ² cardiogeninœ Î i ²Âµ«Œý ŒÉä º q ²¼ µ«Œý Éäq i v ç¼m ² ŒØŒ Cardiogeninq² g p o qg i cardiogeninžg npå Œ ²¼ µ«Œý ŒÉä º Îq ¹ n o p cardiogenin ùx ƒ µ«Œý Œ Éä ÿùœ ƒ¼kü ²¼ µ«Œý Éä ² ² q Œ¼¼mÇ q Œ v q Œ Ùg f 4 õ h [1] Fuster V, Badimon L, Badimon JJ,et al. The pathogenesis of coronary artery disease and the acute coronary syndromes (1).N Engl J Med. 1992;326(4):242-250. [2] Bangdiwala SI, Weiner DH, Bourassa MG,et al. Studies of Left Ventricular Dysfunction (SOLVD) Registry: rationale, design, methods and description of baseline characteristics.am J Cardiol. 1992;70(3):347-353. [3] Bourassa MG, Gurnl O, Bangdiwala SI,et al. Natural history and patterns of current practice in heart failure. The Studies of Left Ventricular Dysfunction (SOLVD) Investigators.J Am Coll Cardiol. 1993;22(4 Suppl A):14A-19A. [4] Kehat I, Kenyagin-Karsenti D, Snir M,et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes.j Clin Invest. 2001;108(3): 407-414. www.crter.org [5] Min JY, Yang Y, Sullivan MF,et al. Long-term improvement of cardiac function in rats after infarction by transplantation of embryonic stem cells.j Thorac Cardiovasc Surg. 2003; 125(2): 361-369. [6] Beeres SL, Atsma DE, van Ramshorst J,et al. Cell therapy for ischaemic heart disease.heart. 2008;94(9):1214-1226. [7] Planat-Benard V, Silvestre JS, Cousin B,et al. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives.circulation. 2004;109(5):656-663. [8] Jackson JD, Zhou G, Kuszynski CA,et al. Induction of chimerism in mice using human MHC class I-mismatched Hoechst 33342 side population donor stem cells.cell Transplant. 2002;11(8):779-785. [9] Orlic D, Kajstura J, Chimenti S,et al. Bone marrow cells regenerate infarcted myocardium.nature. 2001;410(6829): 701-705. [10] Li M, Yu CM, Cheng L,et al. Repair of infarcted myocardium by an extract of Geum japonicum with dual effects on angiogenesis and myogenesis.clin Chem. 2006;52(8):1460-1468. [11] The Ministry of Science and Technology of the Peopleÿs Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals.2006-09-30. fét º y µ. ºp ÍÙgq. 2006-09-30. [12] Jiangsu New Medical College.Shanghai:Shanghai Science & Technology Press.1977:1751. ç Ã.f ±º: ¼[M]. : y Çfx,1977: 1751. [13] Li M,Sung JY,Leung PC,et al. An organic extract of Geum japonicum thumb variant and use thereof[p].wo:2004052381, 2004-06-24. [14] Li M,Sanderson JE,Sung JY,et al.comositions comprising organic extracts of Geum japonicum thunb var and the use thereof[p].wo: 2003043645.2003-05-30. [15] Baksh D, Song L, Tuan RS.Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy.j Cell Mol Med. 2004;8(3):301-316. Ž Èq «Ù RGC grant (2140569) from Hong Kong Government Research Committee (470707)d «h  ì ò À ó ìd n ì ì d ÌrÀ{ ß À jãà ½ Ò ͽÅÍ Ò½í d k  «n t üì d Ë ß Ô v EGJ Åp n ¾ «½ ë Ð ë ò EGJ ½ cardiogenin Åp n ¾ d É cardiogenin Åp ¾ Ìò cardiogenin EGJ Åp ¾ ûd ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH 2539