Microsoft Word 寇建强-5.0-彩图.doc

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1 f zu{ g } 15 í } Çf Journal of Clinical Rehabilitative Tissue Engineering Research January 15, 2011 Vol.15, No.3 ² «ÎÎÂµ«Œ ýéäg±î Œq * 1 2 i 1 i 1 Influence of exogenous hyaluronic acid on chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells Kou Jian-qiang 1,2, Wang Chang-yao 1, Wang Ying-zhen 1 Abstract BACKGROUND: Hyaluronic acid is the most important component of intra-articular synovial fluid and plays a very important role on the cell morphogenesis, but its effect on bone marrow mesenchymal stem cells (MSCs) in the repair of cartilage defects remains unclear. OBJECTIVE: To investigate the role of intra-articular environment to the MSCs by studying the influence of exogenous hyaluronic acid on proliferation and chondrogenic differentiation of rabbit bone marrow MSCs. METHODS: The rabbit MSCs were isolated and cultured in the method of whole bone marrow and adherent culture. The fourth passage of cells were used for the experiment. The experimental group cells were induced by hyaluronic acid solution, with transforming growth factor-β3 induced group served as a positive control. The negative control group was joined regular medium. The features of chondrocytes were identified by toluidine blue staining, immunohistochemistry and RT-PCR to detect the expression of collagen after 7, 14, 21 day s induction respectively. RESULTS AND CONCLUSION: After induced by hyaluronic acid, the speed of cell proliferation slowed down. The cell morphology changed from long spindle to polygonal, oval. The extracellular matrix showed metachromasia with toluidine blue and positive with type collagen immunohistochemical staining. RT-PCR detection also showed the expression of type collagen mrna. All the above showed the characteristics of cartilage cell differentiation. But their expressions were weaker than the positive control group. The results indicated that the exogenous hyaluronic acid can induce the rabbit MSCs differentiate to chondrocytes. But its capacity is weaker than transforming growth factor-β3. Therefore, the intra-articular environment plays a positive role in promoting chondrogenic differentiation of MSCs and supported that hyaluronic acid could be used as a matrix for cartilage tissue engineering. Kou JQ, Wang CY, Wang YZ.Influence of exogenous hyaluronic acid on chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(3): [ ² «º g q É Œq öm så q n ºnp±Î ÎÎÂ µ«œq rq ³²É ² «ÎÎÂµ«Œƒ ö ýéäg±î Œq º ¼j ÎÎ Âµ«Œq n ¹ÎÎ + Éy» ÎÎÂµ«Œ }4x Œnp Í Í ŒØ ² «x ±ämà β3 gâ d Â d Ø» ÉÌp ý} dÿn Œ q Š Ÿ² o ä ört-pcr Œ ŒñŸ² ² «ý Œ ³ Ã nà g Œ «n Œ Œñ o äâ RT-PCR x ŒñmRNAŸ²Â ŸjÇ±Î ŒqÉäg Ÿ² Â d x ² «º ÎÎÂµ«Œý±Î ŒÉäqŒ ±ämà β3q Œ º ¼j ÎÎÂµ«Œý±Î ŒÉä ² n ² «g±î z «ˆn ºÀ ² «ÎÎÂµ«Œ ±Î Œ Éä Œ «doi: /j.issn i i. ² «ÎÎÂµ«Œ ýéäg±î Œq [J].f zu{ g (3): [ 1 Department of Joint Surgery, 2 Central Laboratory, Affiliated , Shandong Kou Jian-qiang, Studying for master s degree, Department of Joint Surgery, Affiliated Hospital of Qingdao University Medical College, Qingdao , Shandong Province, China; Central Laboratory, Affiliated , Shandong kjqwqq@163.com Correspondence to: Wang Ying-zhen, Master, Professor, Master s supervisor, Chief physician, Department of Joint Surgery, Affiliated , Shandong wangyingzhenqd@ 163.com Supported by: the Natural Science Foundation of Shandong Province, No. Y2006C19* Received: Accepted: Î º º Ë} ³ º ±Î g±î Œ p mœ q Éä Œ Ž i q Œ x±î ýq g Ä ~ q±î q µ º Â º ±Î q r v ¼ gº qáè º ±Î p² ±Î ý Ÿ q Œ «x ±Î qž Œ Ä ñ q ØŒ rðg ³²z Žƒ ƒ ±Î º ±Î Œq ( Î ±Î }) oº ±Îq y š Œm ² ±Î ºmg Œ g w Æ } k d ² ISSN CN /R CODEN: ZLKHAH 381

2 { œ 1 Í Þ àè 1984 { x Í Þ Ôd kjqwqq@ 163.com Ì Ô { œ Í Þ f É ú:r318 h u:b ú: (2011) º ( /D Y) 382 n z q º ±Î í xoxtù qð ÎÎÂµ«Œ(bone marrow mesenchymal stem cells BMSCs) ü z º ý Î ±Î Æ Œ x öîî «} É äq Œ [1] ² «º g q É Œq öm så q n rx g±î Œ n p±î q È º BMSCsq h ³ È ˆn ² «º ¼qg mäj É º BMSCs Éäq g ÀBMSCs g uq zä±îqg náè u Œ Âö p200909/ Å ç ÃÂ çãf Í Ÿ º q 6ù 4 Ú Ä Ä Â ƒ«200 g nå Í ù ú SCXK(Ò) Í²z}ürºÙgk [2] g y Ï Ï y ƒ DMEM» (LG-DMEM) Î DMEM» (HG-DMEM) fž (FBS) ˆ Gibco ¹ú ±ämà β3 x Ê ProSpec ¹ú (Dex) m C(VitC) n Œ äsë ¹ú o ä Ïr äsf ¹ú RNA Ïr TAKARA ¹ú RT-PCR Ïr ˆ Biotium ¹ú ˆr À OLYMPUS IX50 PCR y Eppendorf ¹ú ÄŒ y VILBER LOURMAT Í BMSCsqƒ Éy» º q ƒ Ž z Î ŒÎ Ó Î e nÿƒzé 15%FBSq LG-DMEMö À Î À r 50 ml»mf ±± â ˆp37 ƒz É 5%CO 2 ~¼ 3 dýë xý e 1 12~14 dý Œ m x1 2q ä~x ~xýq Œ nÿƒzé 10%FBSqLG-DMEM» ñx Œ gp0 xý gp1p2 BMSCsqmÀ màg q P1P3P5 Œ 2.5 g/lœ q ä x L -1 yp ml 3 ä 3 ~ ² 7~ 9 d x» Âg ± Œ g ± Í mà ² «BMSCsý ±Î ŒÉä }3x m q Œ ä Í Œ Œ Œ x L -1 q yp¹ f ºfµÉ¹ ¼ ˆ Š«È k²qrj f Œr Ã Ég3 Í Â d Â d ¹ ¼q Œ ü² 80% ô ¼» Í Ø ² «(ÿ0.25 g/l² «+ÿƒzé 10%FBS qhg-dmem) Â d Ø ±Î (ÿ10 μg/ltgf-β mol/l Dex+ 50 mg/lvitc+ ÿƒzé 10%FBS q HG- DMEM) Â d Ø» (ƒzé 10%FBS qhg-dmem) ˆp37 ƒz É 5%CO 2 ~¼ Ã ÉÌp ý} Ÿr ˆ À Œ q ä mà À Œffqn Œ ü Âq Œff Ç 40 g/l Šn 1 h Ž À 15 min Ë À 1 Ø1%n Œ pff 2~4 h Ë À ³ Ø ƒzé 95%j ô q À À ý e p n ² f Œ o ä BMSCs ŒñqŸ² ü ÂqBMSCsff Çý Ád 30 min PBS 5 min 3 o ä Ïr lÿsabc RT-PCR Ïr( RNA Ïr RT-PCR Ïr) Œ ¼q RNA ý¼²ÿrt-pcrrgqü Œñ g Ê [3] 5ÿ-AAC ACT GCC AAC GTC CAG AT-3ÿ 5ÿ-CTG CAG CAC GGT ATA GGT GA-3ÿ ¼õ GAPDH g Ê 5ÿ-GAT GCT GGT GCC GAG TAC GT-3ÿ 5ÿ-CCG AAG TGG TCG TGG ATG AC-3ÿ PCRö z P.O. Box 1200, Shenyang cn.zglckf.com

94 3 minè 94 30 s 55 30 s 72 45 s 37f j 72 ² 10 min ö rgÿ2%k Œ ÄŒn GelRed nbiocaptmwäœ ²Ÿ

05g i Cell number ( 10 4 ) 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 Time (d) P1 P3 P5 P7

1 h Ë y Œ ( Œ ³ž Œ}) ºf Ë ý Œ«gu Œ Œ ù u Œ Ä mà g 7~10 d ŒÄ Œ gà v gmà 1 Figure 4

2 Ä æ Î«Â d fu Œn Œ Â 5 Í Â d ŒqŒ «6 dãs Âq À z Ø Í Â d ± Figure 1 Primary culture of bone

h¼¹µ«mà³ Ø ï 24~48 hq ý²³² mà Œ g À u 3~7 dàž ü 2 3 Ø xý Í Â d Œ±Â d mà³ Ã Œƒz² n ² g 4

Extracellular matrix (Blue staining, 100) 6 ŒqŒ «( 100) Figure 2 The third passage bone

3 94 3 minè s s s 37f j 72 ² 10 min ö rgÿ2%k Œ ÄŒn GelRed nbiocaptmwäœ ²Ÿ É n} 21 d RT-PCR É (Quantity One) ºzÉ A ¼õGAPDH q gmrnaÿ² õ nspss 12.0±z²Ÿ É Â²Ÿ É P < 0.05g i Cell number ( 10 4 ) Time (d) P1 P3 P5 P7 Figure 3 Growth curve of bone marrow mesenchymal stem cells 3 BMSCs qmà 2.1 h Ë y Œ ( Œ ³ž Œ}) ºf Ë ý Œ«gu Œ Œ ù u Œ Ä mà g 7~10 d ŒÄ Œ gà v gmà 1 Figure 4 Hyaluronic acid induction 14 d ( 100) 4 ² «} 14 ( 100) 2.2 Ä æ Î«Â d fu Œn Œ Â 5 Í Â d ŒqŒ «6 dãs Âq À z Ø Í Â d ± Figure 1 Primary culture of bone marrow mesenchymal stem cells at 7 d ( 100) 1 ÎÎÂµ«Œñx»} 7 ( 100) ~xý Œ ¼ Ä mà âé q mà 24 h¼¹µ«mà³ Ø ï 24~48 hq ý²³² mà Œ g À u 3~7 dàž ü 2 3 Ø xý Í Â d Œ±Â d mà³ Ã Œƒz² n ² g 4 Figure 5 Negative control group (Toluidine blue staining, 200) 5 Â d (n Œ 200) Figure 6 Extracellular matrix (Blue staining, 100) 6 ŒqŒ «( 100) Figure 2 The third passage bone marrow mesenchymal stem cells at 3 d ( 400) 2 } 3 x Œ~x} 3 ( 400) 2.3 Î«Í 7 dý Œ Ä ŒŒ f µé ŒŒ Ø 7a 14 dxý Œ j ISSN CN /R CODEN: ZLKHAH 383

q ± Œ Ä Œ ù Ø 7b 21 dý j q â 7c rg rü Â d Ÿ² 14 d 21 d Í Â d

Immunohistochemical staining of the experimental group cells

dfk Œñ o ä Â ŒÇj 8 Â d ± Œ Œ ± 9 1: Negative control group;

Figure 10 Results of reverse transcription polymerase chain

201 bp È 384 Figure 8 Extracellular matrix (Blue staining,

Extracellular matrix (Blue staining, 100) 9 ŒqŒ «( 100) ² n z

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4 q ± Œ Ä Œ ù Ø 7b 21 dý j q â 7c rg rü Â d Ÿ² 14 d 21 d Í Â d ù 10b c Â d u Ÿ² c: Induced 21 d c: Induced 21 d Figure 7 Immunohistochemical staining of the experimental group cells induced for different time ( 100) 7 Í ý ü Â o ä 100 Â d 21 dfk Œñ o ä Â ŒÇj 8 Â d ± Œ Œ ± 9 1: Negative control group; 2: Experimental group; 3: Positive control group; a: GAPDH Figure 10 Results of reverse transcription polymerase chain reaction 10 ü RT-PCR É Â d Í Â d mrnaÿ² ÉÌg13.06± ± ±1.02 ü Â ± Å i (P < 0.01) 2.4 RT-PCRÎ«Œq RNA RT-PCR ý kœ ÄŒn 7 d Í Â d 200~300 bpçj± 10a 201 bp È 384 Figure 8 Extracellular matrix (Blue staining, 100) 8 ŒqŒ «( 100) a: Induced 7 d b: Induced 21 d Figure 9 Extracellular matrix (Blue staining, 100) 9 ŒqŒ «( 100) ² n z q º ±Î xoxtù qð Âµ«Œ g z ±Î ²zf nqy Œ [4] ºfBMSCs Ž Ë d pvîîféy ä ƒ Œ } qâµ«œ ñx» Ä äè k d Î» ù BMSCs q±îéä Œ Í Î BMSCsŸ g np±î z u{ gk qy Œ [5-6] BMSCsqÉy» ýéä Íu{q u r ÐnpÉyBMSCsq g «}² y ŒyÉy [7] Í³²¹ÎÎ»+«}² ÉyBMSCs ² Éy»fÇjq «ž Œ e ¼Ã ŸÃô Ä } [8] öjbmscs ñx }1 2x Œ g ùœ ºw Œ }6xxýBMSCsq ±ÎŒ Âƒ ² q P.O. Box 1200, Shenyang cn.zglckf.com

5 ±Î zy Œ g}3~5xbmscs Í nmàg q}4x Œ²Ÿ Í ù n» j ö ²zfq±ÎÐƒg ² Íöj» np³² Âƒ Œ f» Ãv Œ ö Â Œ ± ±Î q [9] ir Í nê» Ê ²ÁÈ ýq Œ Œ ± Œ ± q±î Œ ± Î zqä ÍŸ Œ ƒ»q BMSCs s q ÉäŒ ºf x±ämà Œ ±ämà β mà ñ qbmscséä ±Î [10] ±ämà β ƒβ2 β3 β1ù Ø ³ ²BMSCsý±Î ýéä [11] Íx±ä mà β3 gâ d ý Œn Œ Â s d â Œñ o ä 21 d ±7 d Ø RT-PCR Ÿ ŒñmRNA ÎŸ² n xoº BMSCs q ÉäŒ BMSCs ƒ Éä q±î Œz g Œye Íqƒ»j ² º ¼ Œ ÿ² º ¼qkä É ¼ v h ³ Ä² u{ º ¼² «g n±î Œ B ŒÉ º ±Î «q É [12] º ± qs Î Ø g Ã² «É ö q ü ä [13] ² «ƒ ² Œñ } «É o BMSCs Ëq Œ j x y ² «qu{g Äfp q [14] Johnstone} [15] Šü n² «ŒÍ o y Î iq «ùx ÍBMSCs º¼q±Î ŒÉä yq² «BMSCs ui q ívógtu Í n ² «º ¼qkäj Éy»q BMSCs²Ÿ Éä» dý ²Ÿn Œ Œñ o ä ört-pcr ýqbmscsé ± Î Œg q Œ «Œñ qš xbmscs Éäg±Î Œ d Í f Œñÿ ²ÎpÂ d Â d kçj Â Œ ùœ ž f umà nxö±î Œ y ÌpBMSCsý±Î Œ ýéä º x yq² «kùx BMSCs ý ±Î ýéä d21 d ±Î Œqg «Œñ±7 d Ÿ² Î 3f Â ² «q ÉäŒ ±ämà β3 ² «qg Ã² «É ö q ü äq Maleski} [16] q Ík g² «±Î q ºr É rº ü üé q² «ƒ»qbmscs vä² u{ 4 õ h [1] Liu HW, Chen CH, Tsai CL, et al. Heterobifunctional poly(ethylene glycol)-tethered bone morphogenetic protein-2-stimulated bone marrow mesenchymal stromal cell differentiation and osteogenesis. Tissue Eng. 2007;13(5): [2] The Ministry of Science and Technology of the People s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals fét º y µ. ºp ÍÙgq [3] Park H, Temenoff JS, Tabata Y, et al. Injectable biodegradable hydrogel composites for rabbit marrow mesenchymal stem cell and growth factor delivery for cartilage tissue engineering. Biomaterials. 2007;28(21): [4] Tuan RS, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther. 2003;5(1): [5] Abdallah BM, Kassem M. Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther. 2008;15(2): [6] Hu RY.Beijing: The Publishing House of People s Health. 2006:168. Œ i.jxîy u g [M].äs:t ìmçfx,2006:168 [7] Beyer Nardi N, da Silva Meirelles L. Mesenchymal stem cells: isolation, in vitro expansion and characterization. Handb Exp Pharmacol. 2006;(174): [8] Huo JZ,Chen ZR. Zhongguo Linchuang Yixue. 2005;12(1): Ä Â. Œ~x ÎÎÂµ«Œ ±Î ŒÉäq [J].f g ç,2005,12(1): [9] Duan XJ,Yang L,Zuo ZH.Zhonghua Shiyan Waike Zazhi.2007;24(3): ¾ Àé} nšä» ²Ÿ ÎÎÂµ«Œƒ ±Î»[J] fé Í y,2007,24(3): [10] Roelen BA, Dijke P. Controlling mesenchymal stem cell differentiation by TGFBeta family members. J Orthop Sci. 2003;8(5): [11] Barry F, Boynton RE, Liu B, Murphy JM. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res. 2001;268(2): [12] Yang XL,Liu M.Zhongguo Tengtong Yixue Zazhi. 2006;12(1):47-49.,Ê.² «Îº qu{² [J].f ooç, 2006,12(1): [13] Grigolo B, Lisignoli G, Desando G, et al. Osteoarthritis treated with mesenchymal stem cells on hyaluronan-based scaffold in rabbit. Tissue Eng Part C Methods. 2009;15(4): [14] Wang CT, Lin YT, Chiang BL, et al. High molecular weight hyaluronic acid down-regulates the gene expression of osteoarthritis-associated cytokines and enzymes in fibroblast-like synoviocytes from patients with early osteoarthritis. Osteoarthritis Cartilage. 2006;14(12): [15] Johnstone B, Yoo JU. Autologous mesenchymal progenitor cells in articular cartilage repair. Clin Orthop Relat Res. 1999;(367 Suppl): S [16] Maleski MP, Knudson CB. Hyaluronan-mediated aggregation of limb bud mesenchyme and mesenchymal condensation during chondrogenesis. Exp Cell Res. 1996;225(1): Ž Èq «Ù Þ ¾Ð (Y2006C19) ß m Åp Ôd «h c c Ð Â Ð c Ð ò À c n c c c d Ž Ì c j é od { é d ÌrÀ{ ß À jãà ½ ÒÂ Í½ÅÍ ÒÂ½í d ISSN CN /R CODEN: ZLKHAH 385

Microsoft Word - 会协_2015_1号.doc

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