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1 f zu{ } 18 í } Çf Chinese Journal of Tissue Engineering Research February 12, 2014 Vol.18, No.7 ÎÎ«ŒýuÎ ŒqÉä Â 1 Í 2 Ü ) ( 1 Â{ œ Æ Þ Â{ œ s 1 ÍqËpöj«Éy ÍqÂµ«Œö~xqÂµ«Œfq ŒŒ Ÿ uî Œ Ëü Â«qÎÎ Œ²ŸÉ ü Â«q ü Œˆ ŒqÉä 2 «Éy Íqñx ~x«œf µé Œ ÉäguÎ Œ Œ d ü Â«q ŒˆÉä d i ºÀ Î Œ ÎÎÂµ«Œ uî Œ ³ž Œ «Éy Éy» Éä gè Œ Â«Œ uî Œ Œ»q «Ù y u{ Ê(2012CB966904) g n«éy Íq ÙÎÎfŒ «q ŒgÂµ«Œ ŒÉä g Î Œ ö ±Î Œ ºf«q Œ ÿ ÉäguÎ Œq Œv rq«éy Íq ÙÎÎfŒ«q Œ ÿœ ÉäguÎ Œ n«í ÙñxÂµ«Œ xö³²«15 d ý Í ü Â«qÎÎ Œ ñx Âµ«Œö ü Â«qÎÎ ŒÉÌn ³» öÿ m-csf RANKL»» 9 d ý²ÿ w w u w ñxâµ«œ~xý } 2 xâµ«œég 4 ÉÌn ³» ÿ m-csf» ÿ RANKL» öÿ m-csf RANKL q»» 9 d ý²ÿw w ö u w «Í± qâµ«œ ñxö~x«œqšü u w gâ ñx ~xq«îî Œf ùxéäguî Œq Œ ü Â«q Œ ýw w ö u w ü Â«q Œ Éä m 1989 ± Â{ x À ö Ôd Â{ œ Æ Þ doi: /j.issn [ f É ú:r318 h u:a ú: (2014) ÈÍ Â Í Ü.ÎÎ«ŒýuÎ ŒqÉä[J].f zu{ (7): Osteoclast differentiation of bone marrow adherent cells Chen Jing 1, Ma Ji 2, Sun Fen-yong 1 ( 1 Department of Medical Laboratory, Tenth People s Hospital, Tongji University, Shanghai , China; 2 Department of Central Laboratory, Tenth People s Hospital, Tongji University, Shanghai , China) Abstract BACKGROUND: Generally considered bone marrow cells obtained by adherent method are mesenchymal stem cells, and they can differentiate into osteoblasts, adipocytes and chondrocytes. OBJECTIVE: To explore the differentiation capacity of bone marrow adherent cells into osteoclasts. METHODS: Primary mouse mesenchymal stem cells were obtained using adherent method, and bone marrow cells were obtained through adherence 15 days. Both of them were cultured with normal medium and inducing medium containing m-csf and RANKL. After 9 days, cells were stained by alkaline phosphatase and tartrate-resistant acid phosphatase. The passage 2 mesenchymal stem cells were divided into four groups, which were induced by mock, m-csf, RANKL and m-csf+rankl, respectively. After 9 days of culture, the cells were stained by alkaline phosphatase and tartrate-resistant acid phosphatase. RESULTS AND CONCLUSION: Primary culture of adhered cells was uniform. Alkaline phosphatase and tartrate-resistant acid phosphatase staining of primary and passaged bone marrow adherent cells induced with m-csf+rankl were positive. This result showed that there were cells that could be induced into osteoclasts in the primary and passaged bone marrow adherent cells. Alkaline phosphatase and tartrate-resistant acid phosphatase staining showed differences of adherent cells at different times after the induction, indicating that adherent cells at different times had different differentiation capacity. Subject headings: stem cells; mesenchymal stem cells; osteoclasts; cells, cultured Chen Jing, Studying for master s degree, Department of Medical Laboratory, Tenth People s Hospital, Tongji University, Shanghai , China Corresponding author: Sun Fen-yong, M.D., Department of Medical Laboratory, Tenth People s Hospital, Tongji University, Shanghai , China Accepted: ISSN CN /R CODEN: ZLKHAH 985

2 Funding: the National Key Scientific Research Project of China, No. 2012CB Chen J, Ma J, Sun FY. Osteoclast differentiation of bone marrow adherent cells. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(7): Introduction ÎÎÂµ«Œ ÎÎ¼ yº Î qž ýéä Œq Œ Œýf x ŒÉä z ùx ù Éäg Î Œ ±Î Œ Œ Œ [1-6] ÎÎÂµ«Œ nqéy «Éyy Éy wj Éy } [7] «Éy ÎÎÂµ«Œ Ën Friedenstein} ³²ù Ø»t i h h}îîâµ«œ [8-9] ºwÉy r «É y ~ê d Œ«Âu Í«qÂµ«Œ± äéy [10] Ð u{ öj ³²«Éy ÍqÎÎ Âµ«Œf µé ŒùŸ ÉäguÎ Œ u Î Œ p³ž Œqê ŒÐƒÉ [11] ³ ž Œ Œ«ùŸ ô [12] go² «Éy ÍqÎÎÂµ«ŒfµÉ ŒýuÎ ŒÉäq Œ Í «Â üqîî Œ xö~ x Í}2xÎÎÂµ«Œ²Ÿm-csf RANKL» ³²w w u q w É «ÎÎÂµ«Œý Î uî ýq ÉäŒ 1 Materials and methods Œ mg g Í Âöp Ž r³ Bio-Xf n Í ÍÙg SPF Ÿ C57BL/6 Ù20ù «Ž Æ ÍÙg Â¹ú mr ù ú SCXK ( ) Í²zf Ùgq ˆ}üÙgk Â «Éy Íq ÙÎÎ«ŒýuÎÉä Íg Ï y Ïöy α-mem» fž u w Ïr w w ÏrŒ q ÅÄ Ä CO 2»~ ˆ À Gibco Sigma Thermo Nikon Í ÙÎÂµ«ŒñxÉy» Ø ý ÙÈ Œƒ nƒzé 70%qj min öœ Î ŒÎ ôãÿåqåö PBS Óôe ÎÎ 1 ml ¾ ÎÎ n ž α-mem» ö À n}ˆ ôvî r/miny 10 min ônÿö (ÅÄ ö Ä )ƒzé 10% fž qα-mem» ö Í ÎÎê Œ d qyp24 37 ƒzé 5%CO 2»~f» Âµ«Œq äö~x ñx»²zf 24 h ý Œö» ô npbs 2³ Ø qÿö ƒzé 10% fž qα-mem» xý 3 d 1»ñx Œ ÀŽ90% ú Œ ä 1 2q ²Ÿ~x ÙÎÂµ«Œ¹ õ h[10] n Œy ŒŸÅ gqÿ² }3xÎÎÂµ«Œ Ÿ²CD34 CD45 Ÿ²CD29 CD44 ñxâµ«œq» ñxîîâµ«œég2 ÉÌg{q d m-csföranklšü (m-csf+rankl ) ñx Œ«24 h m-csf+rankl Ø «g25 μg/lqm-csfö 50 μg/lqrankl xý Ø Ï Œ»9 dý xw w ¹ Î Œq x u w Ïr ¹ uî Œ Í 3 ~xâµ«œq» }2xÎÎÂµ«ŒÉg4 ÉÌg{q d m-csf (m-csf ) RANKL (RANKL ) m-csföranklšü (m-csf+rankl ) }2xÎÎÂµ«Œ~x m-csf Ø «g25 μg/l m-csf RANKL Ø 50 μg/l RANKL Šü Ø rü xý Ø Ï Œ»9 dý ²Ÿw w ö u w Í 3 ü Â«qÎÎÂµ«Œq»ö ñ x»f ŒÉg{q d m-csf ö m-csf+rankl Œ« dý Œö» ô ÉÌØ»ñx Œ krü kù Í ü Â«q Œ» 9 dý²ÿw w u w Í 3 g ÎÎÂµ«Œ ñxö ~xîîâµ«œ«œ» ü Â«Íq Œ ýéä Éxx _ ±sÿx nexcel±z² ŸÉ p Â±nt Í P < 0.05 g i 986 P.O. Box 10002, Shenyang

A B C 1 ÙÎÎÂµ«Œq ( 100) Figure 1 Morphology of

Agñx» 3 d qîîâµ«œ Bg~x» 8 d qîîâµ«œ Cg~x» 8 d

Œ ( 1A)»68 dý ŒÀ ² «mà Œ ü Ž90% ú²ÿ1 2~x Œ üž90% ú

9 dýéì²ÿw w ö u w ( 2) {q d ù w w Â q Œ u w Â q Œ

ÎÂµ«ŒÉg4 ÉÌg{q d m-csf RANKL m-csf+rankl 9 dýéì²ÿw

ÿranklqe u w Â q Œ i( 3C) ² ~xýq«œfv ŒùxŸ gu Î Œ 2.

Œ Šü u w Œ 3 d ÎÃý²( 4A B) «Â À «ÍqÎÎÂµ«Œ

dí4 df Í Í( 4C) i A1 A2 B1 A3 B1 B2 B3 C1 C2 C3 D1

primary bone marrow adherent cells from mice after

w ( 40) A3 g{q d w w Â( 100) B1 g{q d u w B2 g{q d

m-csf+rankl C1 g m-csf+rankl w w C2 g m-csf+rankl

3 A B C 1 ÙÎÎÂµ«Œq ( 100) Figure 1 Morphology of mouse bone marrow mesenchymal stem cells ( 100) f Agñx» 3 d qîîâµ«œ Bg~x» 8 d qîîâµ«œ Cg~x» 8 d qîîâµ«œw w ñx»q Œ µé ² Ê«mÀ ~ x Œ ² Ä mà w w Œ qâ Œ 2 Results 2.1 ÅpÎ«h ñx Œ»24 h ý ô Œö» Ø q» ˆ À ù µé Œ ² Ê «mà± Œ ( 1A)»68 dý ŒÀ ² «mà Œ ü Ž90% ú²ÿ1 2~x Œ üž90% ú ˆ À Œ ² Ä mà Œ( 1B) ³²w w Œ qâ Œ( 1C) µéîîâµ«œéäg Î Œ [13] í ØÉyÎÎÂµ«Œ 2.2 Åp Î«ñ x ÙÎÎ Œ«24 hý ö Œ ô { q d m-csf+rankl ÉÌ 9 dýéì²ÿw w ö u w ( 2) {q d ù w w Â q Œ u w Â q Œ m-csf+rankl w w u w µ Â Œ Íq«Œ ÎÎÂµ«Œ ºf q ŒùxŸ guî Œ 2.3 t Åp Î«g Ã¹ ôq Œ «Œ q ñxîîâµ«œ²ÿ~xý }2xÎ ÎÂµ«ŒÉg4 ÉÌg{q d m-csf RANKL m-csf+rankl 9 dýéì²ÿw w ö u w ( 3A B) x4 w w gâ {q d m-csf u w Â q Œ ÿranklqe u w Â q Œ i( 3C) ² ~xýq«œfv ŒùxŸ gu Î Œ 2.4 Å Î«ÐÅ «24 hqñxîîâµ«œ ùé äguî Œ gou{ À«Âý ÍÎÎÂµ «Œ ÿ ÉÌ «15 dqîî Âµ«Œ 9 dý²ÿw w ö u w x em-csf+rankl w w Â q Œ±{q d u w Â q Œ 35 d e w w Â Œ Šü u w Œ 3 d ÎÃý²( 4A B) «Â À «ÍqÎÎÂµ«Œ em-csf+rankl ÍoÎÎÂµ«Œý Î ŒqÉä Œ ÇuÎ ŒqÎÎ Œq ÉäŒ 3 dí4 df Í Í( 4C) i A1 A2 B1 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3 D1 2 ñx»q ÙÎÎÂµ«Œ«Œ» Figure 2 Staining of primary bone marrow adherent cells from mice after induction (1) f A B g{q d A1 g{q d w w A2 g{q d w w ( 40) A3 g{q d w w Â( 100) B1 g{q d u w B2 g{q d u w ( 40) B3 g{q d u w ( 100) (2) f C D g m-csf+rankl C1 g m-csf+rankl w w C2 g m-csf+rankl w w ( 40) C3 g m-csf+rankl w w ( 100) D1 g m-csf+rankl u w D2 g m-csf+rankl u w ( 40) D3 g m-csf+rankl u w ( 100) {q d ù w w Â Œ u w Â Œ m-csf+rankl ù w w u w Â Œ ISSN CN /R CODEN: ZLKHAH 987

A1 C A2 Œ Î u À 40 16 14 12 10 8 6 4 2 a b 0

ÙÎÎÂµ«Œ«Œ» Figure 3 Staining of passaged bone

induction (1) f A g~x«œ 24 w w u w n Žú g{q d

(3) f C g~x«œ À uî Œ {q d m-csf u w Â ÿ RANKL

CD14 CD34 CD45 [15] ÎÎÂµ«Œ ŽqŒ º ý ýéäq Œ

1% ÿ ƒ rð ˆn«Éy y Œ ö owj ²ŸÉy [18] y oî

ü²q~ Û [19] q Í n¹îî«oî q ÙÎÎÂµ«Œ [20-21]

²ºÉä [23] Íf«É y Íqñx ~xxö ü Â«qÂµ«Œ³ ² µé

Kitano} [25] g³ž Œ»² zf ~«² r Ãô e ùx wyqu{ÿ

Ã wy g²q³ž Œ ² ÃgÐƒ ŒqÃ À»Âµ«Œ ~ÿ ³ž Œ â ê Œ

4 A1 C A2 Œ Î u À a b 0 {q d m-csf RANKL m-csf+rankl B1 B2 3 ~x ÙÎÎÂµ«Œ«Œ» Figure 3 Staining of passaged bone marrow adherent cells from mice after induction (1) f A g~x«œ 24 w w u w n Žú g{q d m-csf RANKL m-csf+ RANKL A1 gw w A2 g u w (2) f B g~x«œ À u w n Žú g{q d m-csf RANKL m-csf+rankl B1 g u w ( 40) B2 g u w ( 100) (3) f C g~x«œ À uî Œ {q d m-csf u w Â ÿ RANKL qe u w Â{q d r a P < 0.05 b P < Discussion Âµ«Œ ËÉyŽÎÎf ý Œ Œ ž} f ÉyÇÂµ«Œ [14] Î ÎÂµ«Œ Å³ž q Œ i³ž ŸÅ ñcd11 CD14 CD34 CD45 [15] ÎÎÂµ«Œ ŽqŒ º ý ýéäq Œ [16-17] ÎÎf ÎÎÂµ«ŒëÎÎ Œ q0.001%0.1% ÿ ƒ rð ˆn«Éy y Œ ö owj ²ŸÉy [18] y oî ÎÂµ«ŒqmÀj o Œ Œ ö owj z «d³ Œe «Éy r Œ dz ü²q~ Û [19] q Í n¹îî«oî q ÙÎÎÂµ«Œ [20-21] ÎÎf³ž Œ y Œ«[22] u {Ÿ ÎÎÂµ«ŒùÉ y Œ ³ž Œq º ²ºÉä [23] Íf«É y Íqñx ~xxö ü Â«qÂµ«Œ³ ² µé ŒÉä guî Œh ³ Ù ÎÎfÂµ«Œÿ ƒ d ³ ž ŒôÃ [24] Kitano} [25] g³ž Œ»² zf ~«² r Ãô e ùx wyqu{ÿ }10xqÂµ«Œf Œ CD11 CD45Â ²q ŒŸ g ³ž Œ MSCq»f² Ã wy g²q³ž Œ ² ÃgÐƒ ŒqÃ À»Âµ«Œ ~ÿ ³ž Œ â ê Œ Tropel} [26] Íföj ³ž Œ ù²2f ih ùœ g³ ž Œ g yâs «Œ wy» o18 dqâµ«œ²ÿoÿ öjºf q ŒqCD45Â wy g²q Œ ³ž Œ 988 P.O. Box 10002, Shenyang

A1 C A2 A3 A4 Œ Î u À 40 m-csf+rankl 20 18 16 14 12 10 8 6 4 2 a 1 d 2 d 3 d 4 d 5 d a B1 B2 4 ü Â«q ÙÎÎÂµ«Œ»ý Figure 4 Staining of mouse bone marrow mesenchymal stem cells with different adherent

m-csf+rankl (3) f C g ü Â«Œ À uî Œ xã Â Àw w Â Œ u w Â Œ 3 d 4 d Ã 1 d r a P < 0.

5 A1 C A2 A3 A4 Œ Î u À 40 m-csf+rankl a 1 d 2 d 3 d 4 d 5 d a B1 B2 4 ü Â«q ÙÎÎÂµ«Œ»ý Figure 4 Staining of mouse bone marrow mesenchymal stem cells with different adherent time after induction (1) f A g ü Â«Œ 24 w w u w n Žú g» d A1 g{ q d w w A2 g m-csf+rankl w w A3 g{q d u w A4 g m-csf+rankl u w (2) f B g ü Â«Œ u w ( À 40) n Žú g» d B1 g{q d B2 g m-csf+rankl (3) f C g ü Â«Œ À uî Œ xã Â Àw w Â Œ u w Â Œ 3 d 4 d Ã 1 d r a P < 0.05 g ÎÎÂµ«Œ ÿ ÉäguÎ Œ q Œ n m-csf RANKL º²ŸŠü M-csf gä Î ²uÎ ŒÐƒ Œq ö É äquî ŒRANKL Œ r npuî Œ ºÉä d [27] Íf «ÍqÎÎ Œ²Ÿo öj«íq Œˆf µé Œ ùxÿ guî Œ d ~xq Œfk Œ ùœ µéœéä uî ŒqÐƒ Œ ykùx Âµ«Œ «Éy É y «ü Â«q Œf ýqîî«œw w Â q Œ 3 d ý² u w Â q Œ «3 d 4 d Ã iýö «Â ü ÎÎÂµ«Œöº w«œéäœ ü «3 d ÎÎÂµ«Œ Éä À± dºw«œéäœ ± «3 d ÉyÎÎÂµ«Œ±gü²q Â Íg u{îîâµ«œ o q ö Í u ÇuÎ Œq Œ²ŸÉy ²Ÿ Œ Ÿ q² u{ y ÎÎÂµ«ŒiÂq º Ž «Ã {Þ ÔÂ Ð o Ê { Bio-X ì Ï o d «h Â þãm cþã ÀÚÒ«d ÌrÀ{À À í q d ISSN CN /R CODEN: ZLKHAH 989

6 k «n t 2009 wethical issues in animal experimentationx üì íd ÝÕ ó Ñ ½ ð g ÝÕ ó ÝÕ ó 5b ŠòÎ ì È ì È ãd u k À í À d 4 õ h References [1] Sensebé L, Krampera M, Schrezenmeier H, et al. Mesenchymal stem cells for clinical application. Vox Sanguinis. 2010;98: [2] Cervelló I, Mas A, Gil-Sanchis C, et al. Reconstruction of endometrium from human endometrial side population cell lines. PLoS One. 2011;6(6):e [3] Beane OS, Darling EM. Isolation, characterization, and differentiation of stem cells for cartilage regeneration. Ann Biomed Eng. 2012;40(10): [4] Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bonemarrow osteogenic stem cells: in vitro cultivation andtransplantation in diffusion chambers. Cell Tissue Kinet. 1987;20(3): [5] Nöth U, Rackwitz L, Steinert AF, et al. Cell delivery therapeutics for musculoskeletal regeneration. Adv Drug Deliv Rev. 2010;62(7-8): [6] Holzwarth C, Vaegler M, Gieseke F, et al. Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotentmesenchymal stromal cells. BMC Cell Biol. 2010;11:11. [7] Gronthos S, Zannettino AC, Hay SJ,et al. Molecular and cellular characterization of highly purified stromal stem cells derived from human bone marrow. J Cell Sci.2003; 116(Pt9): [8] Kuci S, Kuci Z, Kreyenberg H, et al. CD271 antigen defines a subset of multipotent stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. Hematologica. 2010;95(4): [9] Zhu H, Guo Z, Jiang X, et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protocols. 2010;5(3): [10] h, é, ñ,}. ÙÎÎÂµ«Œz Éy»ƒ ¹ [J].f zu{ g,2009,13(6) [11] Ê Â,.uÎ ŒÉä ö ú± ³ fç ºÉäq È Í n[j]. ç,2012,16(2): [12] Mori M, Sadahira Y, Awai M. Characteristics of bone marrow fibroblastic colonies (CFU-F) formed in collagen gel. Exp Hematol. 1987;15(11): [13] k,iÿ, È,}. ÙÂµ«Œ» ýmg g u{[j].f Íž,2007,15(3): [14] Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell. 2008; 2(4): [15] Â, é. ü ÎÎÂµ«Œq ýéä[j]. f zu{,2013,17(23): [16], i,,}.îîâµ«œ zu{f q n² [J].f zu{ g,2011,15(49): [17] Cheng L, Qasba P, Vanguri P, et al. Human mesenchymal stem cells support mefakaryocyte and pro-platelet formation. J Cell Physiol. 2000;184(1): [18] Neuhuber B, Swanger SA, Howard L, et al. Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp Hematol. 2008;36(9): [19], Ú, ~,}. ÙÎÎÂµ«Œq» ¹ [J]. f zu{ g,2011,15(10): [20], º, Ö,}.¹ÎÎ«Éy» ÙÎÎÂµ«Œöº Éä[J].f zu{ g,2009, 13(36): [21] Polisetti N, Chaitanya VG, Babu PP, et al. Isolation, characterization and differentiation potential of rat bone marrow stromal cells. Neurol India.2010;58(2): [22] i,ý ±,Ê p. y «Éy» ÎÎÂ µ«œöºmg g [J].f zu{ g,2008;12(34): [23],Êµ,Â,}.ý Î Œ Œ ŒÉäq ÙÎ Âµ«[J].f Íž,2012,20(5): [24] Guo Z, Li H, Li X, et al. In vitro characteristics and in vivo immunosuppressive activity of compact bone-derived murine mesenchymal progenitor cells. Stem Cells. 2006;24(4): [25] Kitano Y, Radu A, Shaaban A, et al. Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells. Exp Hematol. 2000;28(12): [26] Tropel P, Noël D, Platet N, et al. Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow. Exp Cell Res. 2004;295(2): [27] â ì,,iå. ŒÄ Î/KB ƒ g ƒƒ»î uî Œ Ï [J].f zu{ g,2010,14(2): P.O. Box 10002, Shenyang

Microsoft Word - 会协_2015_1号.doc

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