Microsoft Word 张扬5.0彩YJ.DOC

f zu{ } 18 í } 37 2014 09 03 Çf Chinese Journal of Tissue Engineering Research September 3, 2014 Vol.18, No.37 ü» z Œ Œ Î Œqº» 221002) 1 Ê 2 2 ( 1 œ Þ É 221002 2 Þ É s 1 º»ƒ g np Œýù y ŒÉä ŒŽ Éä ŒØŒ Œ ²Ÿ ² qö Îx rgqr 2 ÍqË p } 3 xœ Œ } 2 x Î Œ ü fž z º» ey ŒˆŒ Œý Î ŒÉä ºÀ Œ Œ Œ ÎÉä º» Œ» fž gè Œ Œ Î Œ ŒÉä Œ» Î Œ ÎÎ Œº»ýùx ÎÎ Œý Î ŒÉä Î Œ Œ Œº» ÿkœ ý Î ŒÉä rq Œ Œ Î Œº»ýŒÿý Î ŒÉä Éy º q Œ Œ Î Œ Œ ŒmÀŽ 3 x Î ŒmÀŽ 2 x ²Ÿ º»» ž üég 10% fž º» 5% fž º» º» 14 d º» 7 d ý 2 Œ Œ ÇjµÉ 14 d ý Œ ŒÎ Éä d Î Œr w w   º Œñ ξ mrna Ÿ² Î x 10% fž» g xœ Œ Î Œ ²º»ýùxý Î ŒÉä Î ž»ùx ² n 1984 É 2007 x Þ Ôd Ì Ô Þ É 221002 doi:10.3969/j.issn.2095-4344. 2014.37.018 [http://www.crter.org] f É ú:r394.2 h u:a ú:2095-4344 (2014)37-06003-05 ÈÍ 2014-08-16 Ê. ü» z Œ Œ Î Œqº»[J].f zu{ 2014 18(37): 6003-6007. Co-culture of adipose-derived stem cells and osteoblasts under different conditions Zhang Yang 1, Liu Da-cheng 2, Yang Xiao-ning 2 ( 1 Department of ICU, Xuzhou Medical College Affiliated Hospital, Xuzhou 221002, Jiangsu Province, China; 2 Department of Orthopedics, First People s Hospital of Xuzhou City, Xuzhou 221002, Jiangsu Province, China) Abstract BACKGROUND: After co-culture with osteoblasts, bone marrow stem cells can be induced to differentiate into osteoblasts. Whether adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts or not? OBJECTIVE: To observe whether adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts. METHODS: Adipose-derived stem cells and osteoblasts were isolated from New Zealand white rabbits. Then, passage 3 adipose-derived stem cells were co-cultured with passage 2 osteoblasts in 10% or 5% fetal bovine serum for 14 days. RESULTS AND CONCLUSION: After 7 days of co-culture, some adipose-derived stem cells became round in the two groups. After 14 days of co-culture, adipose-derived stem cells highly differentiated and differentiated cells were similar to mature osteoblasts that were positive for alkaline phosphatase staining and alizarin red staining. The mrna expression of type I collagen and osteocalcin increased in both two group, especially in the 10% fetal bovine serum group. These findings indicate that adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts induced by high-concentration serum culture. Subject headings: adipocytes; osteoblasts; cell differentiation; cell culture techniques Zhang Y, Liu DC, Yang XN. Co-culture of adipose-derived stem cells and osteoblasts under different conditions. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(37):6003-6007. Zhang Yang, Department of ICU, Xuzhou Medical College Affiliated Hospital, Xuzhou 221002, Jiangsu Province, China Corresponding author: Yang Xiao-ning, M.D., Professor, Chief physician, Master s supervisor, Department of Orthopedics, First People s Hospital of Xuzhou City, Xuzhou 221002, Jiangsu Province, China Accepted: 2014-08-16 ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 6003

6004 0 Introduction zî ± zî} Î u{ o ² [1-4] Œ Œ º f Ž ƒ ýéä Œö ¹ Î}~ ²³ gî z qu{ [5] u{ÿ Î Î Œ Î Œº»ýùx ÎÎ Œý Î ŒÉä Œ Œ Îε«Œº r q ŒŸÅ o CDÉ np f ƒ»}~ Œ Œ ùœ xîî⵫œ g q zy Œu{ rð Î Œ Œ Œ ÿ n Í ³ ²Œ Œ Î Œqº» Î ŒŒÿ Œ Œý Î ŒÉä 1 Materials and methods Œ Âö p2012 7 Ž2013 1 ç àçãf Í ÍÙg 3 Ú Ÿ º q 2ù ÄÄ ƒ«2.53.5 kg n ç à ÍÙgf ù úscxk( )2005-0005 Ͳzf Ùg q ˆ}ü2006 y µè q ºp ÍÙgq q [6] ü» z Œ Œ Î Œº»q Íng Ïöy Ïöy DMEM/F-12» fž ˆ Thermo ¹ú Œ q Œñ Œñ vp mg u{ Ù CD44 CD45 ƒ äsë mg ¹ú FITC ˆ IgG äsf mg ¹ú w w Ïr w w Ïr ês mg zu{ RNA Ïr RT Ïr ästian Gen¹ú PCR y ÄŒ ˆ MJ Research¹ú ˆ Alpha Innotech¹ú Œ µ«Œq x2 ml/kgq10% ü Ø ç Å Œ Ç Ãâ ž~ PBS ôã Œ syó Ó 1.02.0 mm 3 Ø 35 ƒz0.1% Œñ 37 ä60 miný Ø }ƒz ÿƒzé 10% fž qdmem/f-12» ä ² Ä 1 200 r/miny 5 min Ø ÿƒzé 10% fž qdmem/f-12» Œ Œ Ä g1 10 9 L 1 yžÿ»»mf ˆpÊ»~»24 h xý 3 d 1 p ˆr À ŒmÀ À ŒmÀ ü²80%90%ý²ÿ ä Ë ~x [7-8] Œ Œq¹ }3x Œx1 10 9 L 1 Œ ypèˆrjfq6 Ø 1.5 mlÿƒz É 10% fž qdmem/f-12» 37 ƒzé 5%CO 2 Ê»~»Ž Œ²80%90% ü nè qpbs 3 5 min 40 g/l Šn 30 min PBS 3 5 min 0.2%Triton X-100² ä25 min PBS 3 5 min ƒzé 5% ˆ Áž 30 min Á Ø Ù CD44 CD45 ƒö 4 ² PBS 3 5 min Ø FITC qp 37 30 min PBS 3 5 min 95%m À À [9-10] Î Œq»ö~x 2 ml/kgq10% ü Š ÅŒ Ø ºq ÉyÈÎ Ã «Î öž~ PBSÀ 3 ÈÎÓ 1 mm 1 mm Ø 0.25%Œ 37 ä Ä 15 miný ä Ø 5 ml 0.1% Œñ 37 f ä Ä 40 miný ä PBSÀ 3 Î 25 cm 2»mf Ø ÿƒzé 20% fž qdmem» ˆp37 ƒzé 5%CO 2»~¼» Î ŒµÉ«nPBS±± 2 ô «q «Œ Ø ¹»» 3 d 1 }6 ÃÎf» Œ m ²80%x ²Ÿ~x» Î Œ Œ Œº» Œ Œ Î Œx1 1q ü Œ y g5.0 10 10 L 1 ÉÌy p6 25 ml»mf ŒÉg10% 5% fž º» 2 2 d 1 ˆr À Œ Œ ä ÉÌp»7 14 d ý Œ ä º» ýqœ Œy p»m»rfxžff RNA w w 14 dýéì Ç2 6 fr jf ÉÌp 7 14 21 28 d Çe Œ 4 y Ä ŒŽ96 Ø 0.2 ml 1%q TritonX-100p4 ² ý dw w r l²ÿ 14 dýéì 2 6 frjf n40 g/l Šn 30 min PBS ýø 2% 1 ml 20 min ˆr À ö± PCR ŒfI Œñ ξ mrnaqÿ ² p Î 7 14 d» n55 mmol/l ¾ ü±ƒ y ý Ä Œ Trizol ϲ Ÿ Œ Œ RNA ²Ÿ ¹ nrt Ïr RNAö± gcdna ý²ÿpcr P.O. Box 10002, Shenyang 110180

A B C D 1 Œ Œ öÿå gqÿ²( 100) Figure 1 Morphology and surface marker expression of adipose-derived stem cells ( 100) f A gñx ŒË ý ôã «Œ ù µé«œ Ê B g} 3 x Œ» 3 d vgº q À g Ê C g o ý Œ CD45 o  Œ Å pž jfq Œ D g o ý Œ CD44 o  Ÿ ²y Œgµ«Œ A B C D 2 Œ Œ Î ä( 100) Figure 2 Morphological changes of adipose-derived stem cells after osteogenic induction ( 100) f A B ÉÌg 10% fž º» 5% fž º» Î 7 d ýq Œ µéœ Œ ö Ê g ³ à ºfx 10% fž g C D ÉÌg 10% fž º» 5% fž º» Î 14 d ý q Œ 10% fž º» Œ Œ Î Éä Î Œ gr 5% fž º» Œ k Î Éä Œ äÿj A B 1 2 3 4 β-actin 400 bp ξ 537 bp Œñ 421 bp 3 Œ Œ ÎÉäq¹ ( 200) Figure 3 Identification of adipose-derived stem cells after osteogenic induction ( 200) f A gœ Œ Î ý w w  À ù Œ Œ B gœ Œ Î ý  À ù q í ¾ 4 ÿ ü q fž» fœ Œ ÎÉä»ý Œñöξ mrna qÿ² Figure 4 Expression of type I collagen and osteocalcin mrna in osteogenic differentiation of adipose-derived stem cells induced by different concentrations of fetal bovine serum 1 5% fž º» 7 d 2 10% fž º» 7 d 3 5% fž º» 14 d 4 10% fž º» 14 d º» 7 14 d 10% fž º» Œñ ξ mrna qÿ² p 5% fž º» Ÿ Œ Œ Î Œ ²Î ž º»ýù ²Œ Œý Î ŒÉä ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 6005

g Ê g ξ Œñ β-actin PCR z 94 È 5 min 94 30 s 52 ² 30 s 72 1 min º²Ÿ30f j ¼ 72 10 min PCRrg n DNA β-actin¼õ q gmrnaÿ² õ g Œ Œ ä Œ Œ Î Œº»ý Œ Œ Î É ä ý7 14 d Œfξ ö Œñ mrnaqÿ² Î Œw w É nspss 13.0±zÉ xx _ ±s Ÿx e ± n t Í ê É ee ± nsnk Í P < 0.05g i 2 Results 2.1 ë Ë Éyq Œ»24 hý ««Œg Œ µé«œ ŒË ý «ŒŸôà û Âq À Œ g º qà Ä gmà Œ ü² 80%90% 3x Œ g mà ŒÀ z»( 1A B) o x ŒŸÅ ñ CD44  Ÿ ²y Œgµ«Œ CD45  Œ Å pž jfq Œ( 1C D) ùxë ¹ Éyq ŒgŒ Œ 2.2 Œº» 7 dý µéœ Œ ö Ê g ³ à ºfx10% fž g ( 2A B) º»14 dý 10% fž º» Œ Œ Ÿj Î Œ g ² 5% fž º» k Î Œ Œ äÿj( 2C D) 2.3 n Æ ó À ³ Ç Î 14 d 2 Œw w gâ ˆr À ù Œ g Œ g Ø 10% fž º» qâ Œ ±5% fž º» Î 14 d 2 Œ  À ù 10% fž º» Œf¾ ±5% fž º» dƒz ( 3 Ÿ1) 6006 Ê 5 -CAT GAG AGC CCT CAC A-3 5 -AGA GCG ACA CCC TAG AC-3 5 -GGC AAA CAT GGA AAC CG-3 5 -TCA AGG AAG GGC AAA CG-3 5 -GTG GGG CGC CCC AGG CAC CA-3 5 -CTT CCT TAA TGT CAC GCA CGA TTT C-3 Ÿ 1 Œ Œ Î ü rqw w Table 1 Acitivity of alkaline phosphatase at different phases of osteogenic differentiation of adipose-derived stem cells (x _ ±s, n=4, 10 King s unit/l)»ý Â( ) 10% fž º» 5% fž º» 1 0.812 1±0.098 0 a 0.351 4±0.033 9 2 1.681 6±0.143 3 ab 0.576 5±0.074 0 3 1.076 6±0.377 9 ab 0.430 6±0.147 3 4 0.879 0±0.117 0 ab 0.387 6±0.055 7 Ÿ 5% fž º» r a P < 0.05 Ð Â r b P < 0.05 2.4 n, Ê œ mrna { ö± PCR x Î 7 14 dý 2 Œ Í Œñ ξ mrnaqÿ² dv e Ÿ² à  v Ì r Í g vÿ²rg ŒñmRNAqŸ² f vü Â Í Œñ ξ mrnaq Ÿ² p d ( 4) 3 Discussion Î q~ o â ŽƒÎ ƒîöt Îz } o qo ŽƒÎ È Î~ o ƒî t κ ñ o ö ~ Ž «xö rö o ~ }ÉȺ n  [11] ÃsÎ z ö kgî q o Î z n z qñk uíº Î ²Î¼mŒ qî xg Íq zîœ ~ oq º qmgr q Œxömgz ÌnÎ z»qt Î uù ÅzqÎ dù Ä ö Í yk që öøœ q ² o qö v y Œ k q dqî xgÿuíç zî ± zî} Î u{k o ² [2-4,12] rðî zu{ qy Œ ÎÎ Œ ÎÎ Œ Í ÂÍ Œ Íu{ög nqä Œ Œ üîî Œ pf œ Œ Œ ÿ ÎÎ Œü º ýéä Œ g qy ŒŒ Œqmg g Îε«Œr [13] Zuk} [14] 2001 vtœ f o ýéä Œq «Œ g ùxý Î Œ ±Î Œ ö ŒÉäqŒ Œ Œ Îε«Œr º x ~ f Ž Œ«Œ pƒ»» z~ê ƒ Œÿ f i Î ŒŸ z Ÿ Î ö k³ ÁÈ [15] Œ Œ ùœ xîîâ P.O. Box 10002, Shenyang 110180

µ«œ g q zy Œ z g â y Œ ±ƒ Œ Œ g qy Œ rð gî ±Î zu{q º»q q~ q z ˆŒ Œý Î ŒÉä ²y ùœ g Íf n o Œ ŒŸÅÉ x ŒŸÅ ñcd44  Ÿ ²y Œgµ «Œ CD45  Œ Å p ž jfq Œ ùxë ¹ Éyq Œg Œ Œ Î Œqg ØŒ ü É Î «²Î «uä Î ¾y Î ŒÉ qw w n z Œñ «quä²z rð w w q g Î Œ¹ zº ØŒg qg º» 14 dýe Œqw w ¾ä  ³² 728 dqw w ¾y ä²ÿé öj w w 714 d² ý²  ¾y 728 d ² Û dâ¾ rº Îp d d r Í ¾ä ƒz Í 7 14 d Î Œ q Œñ ξ mrnaÿ ²qö± PCR p d n jo Œ Œý Î Œq Éä ü k Î Œ º ²Œ Œý Î ŒÉäq n Œ «q¾ä Î ŒÉä qg ƒ» z Î Œrm¾ä ¹ Î Œq ƒ ¾ qg ºŒ fq ¾y Óü x¾ zqµƒ w w qÿ² Î ŒqÉä d rº Œñ Î «q g Éi Œ³² q ˆÅ qw ä Ø Œ q«qü ù ù äna + /H + r ² Î Œq  Éä ù ² Î [16-17] ξ n Î Œü É ö Î Œ qg q g u{öjî¾ Îo x ùœ ŽÎ fîœ q²zqñ [18] ² Íš ƒ ØÌn Î Œ Œ Œº» Œ Œý Î ŒÉä ýq Î ŒŒÿ ±ƒ ²Ÿ qmà Œÿ ƒ¼²ÿ» ² ² º Î Åq Ùg Íu{ «h Úe ò Âc À d ÌrÀ{ À À í q d k «n t «Þ Á 2006 w Çx d è Í ÀÉ 2 ½ 2 ½ Í à ê Å ÍÍtdéÅÍ ÀÉ 2 ½ 2 Í É ð Ó ê ÍÍtd u k À í À d 4 õ h References [1] Yu H, VandeVord PJ, Mao L, et al. Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization. Biomaterials. 2009;30(4):508-517. [2] ëýd,µ é,ê,}.± Œ zîqë Íu{[J].fé y,2010,33(2):143-147. [3] Á, ì,.mg Î öº Œ z[j].f zu{ g,2010,14(16):2963-2966. [4],,. zät Î Î q Íu{ [J].f u y,2009,17(16):1258-1261. [5] Tobita M, Orbay H, Mizuno H. Adipose-derived stem cells: current findings and future perspectives. Discov Med. 2011; 11(57):160-170. [6] fét º y µ.ºp ÍÙgq. 2006-09-30. [7] Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13(12): 4279-4295. [8] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2):211-228. [9] Prichard HL, Reichert WM, Klitzman B. Adult adipose-derived stem cell attachment to biomaterials. Biomaterials. 2007;28(6): 936-946. [10] Rodriguez AM, Elabd C, Amri EZ, et al. The human adipose tissue is a source of multipotent stem cells. Biochimie. 2005; 87(1):125-128. [11] Œ i.jxîy u g [M].äs:t ìmçfx,2006: 151-153. [12] Yu H, VandeVord PJ, Mao L, et al. Improved tissue-engineered bone regeneration by endothelial cell mediated vascularization. Biomaterials. 2009;30(4):508-517. [13] De Ugarte DA, Morizono K, Elbarbary A, et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs. 2003;174(3):101-109. [14] Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2):211-228. [15] Halvorsen YC, Wilkison WO, Gimble JM. Adipose-derived stromal cells--their utility and potential in bone formation. Int J Obes Relat Metab Disord. 2000;24 Suppl 4:S41-44. [16] Mizuno M, Kuboki Y. Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen. J Biochem. 2001;129(1):133-138. [17] Shi S, Kirk M, Kahn AJ. The role of type I collagen in the regulation of the osteoblast phenotype. J Bone Miner Res. 1996;11(8):1139-1145. [18] Ducy P. The role of osteocalcin in the endocrine cross-talk between bone remodelling and energy metabolism. Diabetologia. 2011;54(6):1291-1297. ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 6007