f zu{ } 17 í } 14 2013 04 02 Çf Chinese Journal of Tissue Engineering Research April 2, 2013 Vol.17, No.14 doi:10.3969/j.issn.2095-4344.2013.14.003 [http://www.crter.org] ¾ Ü Â Ä. [J].f zu{ 2013 17(14):2488-2494. o v qtî öm q2 ± Îε«Œ* ¾ Ü Â Ä Î Ú ÒÚ Þ È 350025 s 1 Í n y ü«œ}² ²Ÿ ŒqÉy}² Ãs~x q Ø ÍÎ qîî⵫œ 2 Šü n Œ Œy ŒŸÅ gö ý Éä Œ²Ÿ¹ Í o Í ŒgÎε«Œ 3³² o ±ƒ Ž q r ± À 4 ÍÍ o o ±ƒg ±ƒ rq tî öm q 2 Îε«Œ f ± ýqîî⵫œù ØŸ²tÎ öm q 2 öw w ºÀ Œ ÎÎ Œ o ±ƒ tî öm q 2 Îε«Œ ± Ù w w MTT z y Œ Î r Œ f Îε«Œ gî ±ÎË ö² qy Œ ͺ rq É tî öm q 2 ± q Áˆ Ù(SD Ù)Îε«Œq Éy ä SD ÙÎε«Œ ƒ ³² o ±ƒv tî öm q 2 ± Îε«Œ É̳² À Ÿ² Àö q«± ýtî ö m q 2 qÿ² w w ¹ Î ö MTT tî öm q 2 ± ÎΠµ«œq v SD ÙÎÎ gféy»q Œ g ug gmà Œy ö ÈÉäŒ ¹ }üîî⵫œqg ± tî öm q 2 ý Îε«ŒŸ²tÎ öm q 2 w w MTT ± tî öm q 2 ý Îε«Œ Œ (P < 0.05) tî öm q 2 ± ÎΠµ«œýùx Î Ÿ²tÎ öm q 2 w w ƒ ²Îε«Œq n» 1971 2003 Î Ú Ú { š x óu Þ Ôd chenghuiyin@ hotmail.com f É ú:r394.2 h u:a ú:2095-4344 (2013)14-02488-07 2012-07-17 º 2012-08-26 (20120417013/DeS) Adenovirus-mediated human bone morphogenetic protein 2 gene transfects bone marrow mesenchymal stem cells Yin Cheng-hui, Qiu Jun-qin, Zeng Zhao-xun, Chen Zong-xiong Department of Orthopedics, Fuzhou General Hospital, PLA Nanjing Military Area Command, Fuzhou 350025, Fujian Province, China Abstract BACKGROUND: Bone marrow mesenchymal stem cells as the seed cells for repair of bone and cartilage trauma and degeneration have been paid increasing attention. 2488 P.O. Box 1200, Shenyang 110004
Yin Cheng-hui, M.D., Associate chief physician, Department of Orthopedics, Fuzhou General Hospital, PLA Nanjing Military Area Command, Fuzhou 350025, Fujian Province, China chenghuiyin@hotmail.com Supported by: the Youth Science and Technology Talent Innovation Program of Fujian Province, No. 20055075* Received: 2012-07-17 Accepted: 2012-08-26 OBJECTIVE: To investigative the effects of human bone morphogenetic protein 2 gene transfection on Sprague-Dawley rat bone marrow mesenchymal stem cells. METHODS: Sprague-Dawley rat bone marrow mesenchyal stem cells were in vitro isolated, purified and amplified. Adenovirus-mediated human bone morphogenetic protein 2 was transfected into bone marrow mesenchymal stem cells. CD90 and CD45 expression levels were tested by flow cytometry. The successfully packaged virus was transfected into bone marrow mesenchymal stem cells and expression of human bone morphogenetic protein 2 gene was confirmed by enhanced green fluorescent protein expression under the fluorescence microscope. Enzyme linked immunosorbent assay was performed to monitor the expression levels of human bone morphogenetic protein 2 and alkaline phosphatase in mesenchymal stem cells. The effect of human bone morphogenetic protein 2 on the proliferation of bone marrow mesenchymal stem cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells were successfully harvested from bone marrow of sprague-dawley rats and identified by flow cytometry. After primary culture for 710 days, cultured cells displayed typical fusiform shape and the growth status was like cobblestones or whirlpool under light microscope and could be differentiated into osteoblast-, adipocyte- and neuron-like cells in vitro. After transfection by human bone morphogenetic protein 2 gene, bone marrow mesenchymal stem cells expressed human bone morphogenetic protein 2 and alkaline phosphatase. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that after transfection by human bone morphogenetic protein 2 gene, bone marrow mesenchymal stem cells exhibited a stronger proliferation capacity (P < 0.05). These findings suggest that human bone morphogenetic protein 2 gene-transfected bone marrow mesenchymal stem cells can successively express high level of human bone morphogenetic protein 2 and alkaline phosphatase, indicating that human bone morphogenetic protein 2 gene can significantly promote the proliferation of bone marrow mesenchymal stem cells. Key Words: stem cells; bone marrow-derived stem cells; adenovirus vector; human bone morphogenetic protein 2; bone marrow mesenchymal stem cells; gene transfection; rats; alkaline phosphatase; MTT assay; gene engineering; seed cells; bone defects; provincial grants-supported paper; stem cell photographs-containing paper Yin CH, Qiu JQ, Zeng ZX, Chen ZX. Adenovirus-mediated human bone morphogenetic protein 2 gene transfects bone marrow mesenchymal stem cells. Zhongguo Zuzhi Gongcheng Yanjiu. 2013;17(14):2488-2494. 0 Îε«Œ pîîfº ÈÉä Œq Œ [1] ² q j ùéäg±î Œ Î Œ Œ x Œ} [2] z z k qy Œ dº ²³ oñ Œz Ÿ² º ÈÉä Œq Œ º gî ±ÎË ö² qy Œ ͺ Î q2 κÀg«[3-4] õ v Œ y Éä Í Œ r} Êqmg²z ͳ² z o v qî öm q2 ± ÎΠµ«Œ u{º µ«Œq g² oî qùg Í u 1 Œmg Í Âö p2010 4 Ž2011 3 ¾ês¾çy Ã Í Í qmg ¹q gbsl-2 gr Í o ±ƒ tî öm q2 o Ÿ²±ƒg ¾ Üpy çã Í ¹ nadmax o â ( mgy ¹ú)â ÍÙg Ÿ Ä SD Ù 24ù 3 Ú ƒ«5080 g n ¾ês¾ çy çãùg Íf ù SYXK-(¾)2007-036 Ê»1 ý² Œ Í Í²zf Ùgq ˆ}üç k  ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 2489
2490 g Ïöy Main experimental reagents and instruments: Ïöy DMEM/F12» FBS Ùw w Elisa Ïr tî öm q 2 ELISA Kit Ïr Trizol RNA Ïr qrt-pcr Ïr O Ï IX51 mg ˆ À Q-IMAGING udr SZX2-ILLB À EPICS XL Œy TDZ5-WS çnú y Hyclone ¹ú 鈹ú invitrogen ¹ú òá smg ¹ú Olympus ¹ú Coulter ¹ú À Åy yÿ ¹ú Í SD ÙÎε«Œ»ö¹ n¹îî y ü«œ}² Éy»SD ÙqÎΠµ«œ [5] 710 d«œ(ñx Œ) À ê ý 1 3 ²Ÿ~x» ~xž}3x Œ n ³² Œyö ý ÉäŒ ²Ÿ¹ o ±ƒv tî öm q2 ± ÙÎΠµ«Œ ³² üo (60 80 100 120) ÙP3qÎε«Œ po ± 4872 hý Àö À ÉÌ qÿ² À ŒmÀ À ² ~qo (100)²Ÿ± ÙÎε«Œ RT-PCR rq Trizol RNA Ïrö qrt-pcr Ïr rq 1%kŒ n ¹ RT-PCRrg Š o  Í(ELISA) tî öm q2öw w qÿ² ˆtÎ öm q2± { o ± ö ± ÉÌp± ý3 6 9 12 d Œ w w Elisa Ïr MTT tî öm q2 ± Îε«Œq tî öm q2± {o ± ö ± ü ÉÌ 6f Ÿ ÉÌp y ý16 d²ÿmtt n¹žù y490 nm (A 490 ) ü Í 3 x xspss 17.0 ±z k xx _ ±sÿx ü ± nt Í g ÙÎε«ŒqÉy» ¹ x üo ± Îε«Œq Ÿ²q RT-PCR¹ rq q Š o  Í(ELISA) tî öm q2 qÿ²öw w q MTT Î öm q2 Îε«Œ q É nx _ ±sÿx ü  ± nt Í ³²SPSS 17.0 ±z²ÿ k n}p n É ±  xp < 0.05Ÿx i 2 2.1 { Åp ë Ç ÙÎε«Œq Œ ˆ À ù ñx ŒË y É ž Œr p 1A 3 dý Ãô «Œ ù Œ u ö 1B 710 dý Œg d Ä u g Ê mà 1C ~xý Ãs~x q Ø ù Í Î qîî⵫œ 1D A ñx ŒË y É ž Œrp C 710 d ý Œg d Ä u g ÊmÀ Figure 1 B 3 d ý Ãô «Œ ù Œ u ö D ~Ž} 3 xqî Îε «Œ ~ xgîî⵫œ 1 ü  q ÙÎε«ŒmÀ g ( 100) Morphological characterization of rat bone marrow mesenchymal stem cells under different time periods ( 100) Œy Îε«ŒqŸÅ g Œy P3 Îε«ŒqŸÅ gq g CD90 Œë99.7% 2A CD29 Œë 99.4% 2B CD45 Œë1.78% 2C CD34  Œë0.96% 2D ù ³² y Éy «Œ}²»ù Î qîî⵫œ P.O. Box 1200, Shenyang 110004
Îε«Œ Î 3 ývon Kossa uä 3A Îε«Œ 48 h Œ o NSEŸ²Â 3B ÎΠµ«Œ Œ 2 ý O Œ ¼ù q Œ 3C A CD90 C CD45 CD90  Œë 99.7% CD29  Œë 99.4% CD45  Œë 1.78% CD34  Œë 0.96% ºf CD90 Œ Î Ÿ² Îε«Œ qÿå 2 ÙÎε«ŒŸÅ g B CD29 D CD34 Figure 2 Flow cytometry of surface markers of rat bone marrow mesenchymal stem cells 2.2 Åp { ã ÉÌx üo ± P3x µ«œ 3 dý Ÿ² ù o =80 Ÿ² 4A o =100 ± ³ Ø ù µéîî⵫œ Ÿ² q 4B o = 120 kù µéîî⵫œÿ² q 4C ˆr À öj ŒmÀg ± r Œ o Œq 4D ÙÎε«Œq ýéäœ 3 A o =80 u Ÿ²( À) B o =100 ù µé q ( À) A Œ Î 3 ý Von Kossa uä (~ ) B Œ x 48 h Œ o NSE Ÿ²Â (~ ) C Œ Œ 2 ý O Œ ¼ù q Œ (~ ) Œ²Ÿ Éä ºùÉäg Î Œ Œ Œö x Œ }üîî⵫œq ýéä Œ 3 ü ÂýÎε«Œq ýéäœ ( 100) Figure 3 Detection of multipotent differentiation of bone marrow mesenchymal stem cells after induction for different time periods ( 100) C o =120 À kù µé q D o =120 ˆr À öj ŒmÀg ± r Œ o Œq ± ýqîî⵫œœ öÿ² o =100 ŒmÀ r ä Œq ±± ùx ÍÄ 4 o g 80 100 ö 120 Îε«Œ qg ( 100) Figure 4 Morphology of transfected bone marrow mesenchymal stem cells when the multiplicity of infection was 80, 100 and 120 ( 100) 2.3 RT-PCRÆg ¾ GAPDHg168 bp 1%k Œ ÄŒn pr ƒˆçjrq 5 ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 2491
600 bp 500 bp 400 bp 300 bp 168 bp 200 bp 100 bp 1 600 bp DNA marker 600 bp 500 bp 400 bp 300 bp 200 bp 100 bp 2 GAPDH 5 RT-PCR GAPDH Figure 5 Electrophoresis analysis of the glyceraldehyde-3- phosphate dehydrogenase in reverse transcription-pcr products o =100± Œ72 hýqîîâµ «ŒŸPT-PCR 1%kŒ ÄŒ n 40 min ÄŒ y 6 2.4 î œ (ELISA)Æg ~ 2 { xü rq qqé 7 q m ö Î t 2 (ng/l) 2 000 bp 1 000 bp 500 bp 250 bp 150 bp 140 120 100 80 60 40 20 0 tî öm q 2 ± 1 500 bp 1 2 kb Mark 2 tî öm q 2 o =100 ± Œ 72 hý Îε«ŒÎ Ÿ²tÎ öm q 2 q 6 RT-PCR tî öm q 2 Figure 6 Electrophoresis analysis of human bone morphogenetic protein 2 in reverse transcription- PCR products {o ± ± tî öm q 2 ± tî öm q 2 Ÿ² Î p{o ± ö ± 7 ELISA Îε«Œ± 3 d ýtî öm q 2 qÿ² Figure 7 Detection of human bone morphogenetic protein 2 expression in bone marrow mesenchymal stem cells after gene transfection for 3 d by enzyme linked immunosorbent assay tî öm q2± {o ± ö ± tî öm q2éìg(123.62±3.12) (32.51± 2.83) (38.34±2.76) ng/l ù tî öm q2± tî öm q2ÿ² Îp{o ± ö ± i(p < 0.05) {o ± ö ± iâ i(p > 0.05) 2.5 î œ (ELISA)ÆgÆ ó ½ tî öm q2 o Ÿ²±ƒ± w w Îp{o ± ö ± i(p < 0.05) {o ± ö ± iâ ± i(p > 0.05) Ÿ1 Ÿ 1 Œ tî öm q 2 ± {o ± ý ü Âw w qÿ² Table 1 Alkaline phosphatase activity in each group at different time periods (x _ ±s, 16.67 nkat/l)  3 d 6 d 9 d 12 d tî öm q 2 ± 78.68±24.34 a 105.69±23.17 a 115.86±24.00 a 124.02±26.34 a {o ± 25.51±13.00 38.84±11.00 42.00±12.34 49.34±13.50 ± 28.01±17.34 34.17±15.34 39.01±18.17 56.35±18.34 ºwe ± a P < 0.05 tî öm q 2 o Ÿ²±ƒýw w Îp{o ± ö ± {o ± ö ± iâ i 2.6 MTT Æg ~ 2 Åp Ÿ2 Ÿ 2 Î öm q 2 {o ± Îε«Œ q Table 2 Effect of human bone morphogenetic protein 2 gene transfection and empty virus transfection on proliferation of bone marrow mesenchymal stem cells  1 d 2 d 3 d 4 d 5 d 6 d tî öm q 2 ± 0.136 a 0.197 a 0.342 a 0.465 a 0.608 a 0.682 a {o ± 0.122 0.182 0.288 0.386 0.491 0.534 ± 0.124 0.190 0.301 0.398 0.504 0.583 ºwe ± a P < 0.05 tî öm q 2 ± ý Îε«Œq Œ Îp{o ± ö ± {o ± ± iâ 2492 P.O. Box 1200, Shenyang 110004
tî öm q2 ± ý Îε«Œq Œ Í tî öm q2 o Ÿ²±ƒ± Îε«Œq Œ Îp{o ± ö ± (P < 0.05) {o ± ± ± i(p > 0.05) 3 3.1 Îε«Œ gk qy Œ º x mg g Ž p ƒ ƒ p» Éy äö ~xœ ŽŽƒ È ñ º qmg ¹ º ýéäœ ²np y q iƒ x Î ³²± rq ƒ¼ Ÿ² [6-8] Îε«Œqÿ ƒ ugîîê Œq0.001%0.01% [9] ²²² Íu{Ä ³²ñx ~x» º ³²Éy» jƒ ºŒ Ø nqºà Í n y ü«œ}² d Œ«Œ qñk²ÿéy}² Ãs~x q Ø ù ÍÎ qîî⵫œ ~x r ² ïå ~x»ýîî⵫œ ³ ² Ã Í gîî⵫œq ƒ Œ Ãs Ú ² à ² Ú Ù²Ÿñx [10] ͳ²Šü n Œ Œy ŒŸÅ gö ý Éä Œ²Ÿ¹ [11] À ù Í Œ g ugmà }üîî⵫œ màg ¼ Œ²Ÿ Éä ºùÉäg Î Œ Œ Œö x Œ CD90qÎ Ÿ² Í Îε«ŒŸÅ q [12] dîîâ µ«œœÿ²cd44 CD90 CD106}ŸÅ Ÿ²³ž ŒŸÅ CD34 CD14 CD45 [13-14] Í CD90 i99.7% CD45 iu 1.78% ³ Ž ÍÉy»q Œ}ü Îε«Œqg 3.2 ~ 2¾ { Åp mà Î q ±ä mà β}kù º ÎÉä [15-16] ºfÎ ö m q2 Ÿ¹ grð qî [17] Î ²zf ³² É É Œö ŒÂ²Ÿ ~ ŒÉ m Î üfö s q n üq±ƒ rüqå Œq± i ü rüq±ƒ üqå Œq± ir ü [18] ͳ²}² ~qo Í q± i o ío o Œ q ¼³² Îo ùx Œ o ± q ² α i ²Îqo ~ ± irm«å Œ } Ͳnq o ±ƒ Ž q r ± À Í ˆo60 80 100ö120qo öjo =100 Îε«ŒmÀ r ä Œq ±± 90%x ŒÇj 72 h ¼íù µé ŒŸ² q ± ýîî⵫œœ öÿ² ÍqÄ ³²ELISA Ïr Œ fqtî ö m q2öw w ÇtÎ öm q2 ± Îpºw (P < 0.05) ± ýîî⵫œœîÿ²rq q dº q Î 3.3 ~ 2¾ Åp MTT rð Í nqƒ Œ màq ùnpîî⵫œqƒ }² Ä Œ Î [19-21] Í ± tî öm q2 Îε«Œ q ² n d Îε«Œq Ì g ƒ¼ùg fq² u{ z ÍÌn o ±ƒg ±ƒ rq tî öm q2 Îε«Œ f ± ýqîî⵫œù ØŸ²tÎ öm q2öw w MTT Ÿ tî öm q2 ± ýqîî⵫œ Œ ù Ù gƒ¼ f² u{ «Ù È ÞÁ du (2005J075)d «h ß Â ß c ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 2493
d ÌrÀ{ ß À jãà ½ Ò ͽÅÍ Ò½í d k À üì q d u À ð k ß À íá d 4 õ h [1] Cheng SL,Lou J,Wright NM,et al.in vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene. Calcif Tissue Int.2001; 68(2):87-94. [2] Czernik M, Fidanza A, Sardi M, et al. Differentiation potential and GFP labeling of sheep bone marrow derived mesenchymal stem cells. J Cell Biochem. 2012 Aug 6. doi: 10.1002/jcb.24310. [3] Chang SC, Chung HY, Tai CL,et al. Repair of large cranial defects by hbmp-2 expressing bone marrow stromal cells: comparison between alginate and collagen type I systems. J Biomed Mater Res A. 2010;94(2):433-441. [4] Han D, Li J, Guan X. Ectopic osteogenesis of hbmp-2 gene-transduced human bone mesenchymal stem cells/bcb.connect Tissue Res. 2010;51(4):274-281. [5] 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):1460-1469. [6] Riew KD, Lou J, Wright NM,et al.thoracoscopic intradiscal spine fusion using a minimally invasive gene-therapy technique. J Bone Joint Surg Am.2003;85(5):866-871. [7] Gu HL, Liu L, Lv G, et al. Zhonghua Shiyan Waike Zazhi. 2007;24(3):368. È,Ê,ýË,}. Ad-tÎ q-2± t ÂrÎ Œ Œ «x q [J].fé Í y,2007, 24(3):368. [8] Yao Y, Zhang F, Wang L, et al. Lipopolysaccharide preconditioning enhances the efficacy of mesenchymal stem cells transplantation in a rat model of acute myocardial infarction. J Biomed Sci. 2009;16:74. [9] Wang JC, Kanim LE,Yoo S, et al.effect of regional gene therapy with bone morphogenetic rotein-2-producing bone marrow cells on spinal fusion in rats.j Bone Joint Surg Am. 2003;85(5):905-911. [10] Roth CM, Sundaram S. Engineering synthetic vectors for improved DNA delivery: insights from intracellular pathways. Annu Rev Biomed Eng. 2004;6:397-426. [11] Park BW, Kang EJ, Byun JH,et al. In vitro and in vivo osteogenesis of human mesenchymal stem cells derived from skin, bone marrow and dental follicle tissues. Differentiation. 2012;83(5):249-259. [12] Kisiel AH, McDuffee LA, Masaoud E, et al. Isolation, characterization, and in vitro proliferation of canine mesenchymal stem cells derived from bone marrow, adipose tissue, muscle, and periosteum. Am J Vet Res. 2012;73(8): 1305-1317. [13] Gonçalves Fda C, Paz AH, Lora PS, et al. Dynamic culture improves MSC adhesion on freeze- dried bone as a scaffold for bone engineering. World J Stem Cells. 2012;4(2):9-16. [14] Moscoso I, Rodriguez-Barbosa JI, Barallobre-Barreiro J,et al. Immortalization of bone marrow-derived porcine mesenchymal stem cells and their differentiation into cells expressing cardiac phenotypic markers. J Tissue Eng Regen Med. 2012;6(8):655-665. [15] Murray SJ, Santangelo KS, Bertone AL.Evaluation of early cellular influences of bone morphogenetic proteins 12 and 2 on equine superficial digital flexor tenocytes and bone marrow-derived mesenchymal stem cells in vitro. Am J Vet Res. 2010;71(1):103-114. [16] Han D, Li J, Guan X.Ectopic osteogenesis of hbmp-2 gene-transduced human bone mesenchymal stem cells/bcb.connect Tissue Res. 2010;51(4):274-281. [17] Shingeno K,Nakamura T.Regenerative repair of the mandibler using a collagen sponge containsing TGF-β1.The international of artificial organs. 2002;25(11):1095-1102. [18] Yan MN, Dai KR, Tang TT,et al.reconstruction of peri-implant bone defects using impacted bone allograft and BMP-2 gene-modified bone marrow stromal cells. J Biomed Mater Res A. 2010;93(1):304-313. [19] Real RP, Ooms E, Wolke JG,et al. In vivo bone response to porous calcium phosphate cement. J Biomed Mater Res A. 2003;65(1):30-36. [20] Ten Dijke P, Fu J, Schaap P, et al. Signal Transduction of Bone Morphogenetic Proteins in Osteoblast Diferentiation. J Bone Joint Surg Am. 2003;85(3):34-38. [21] Mamalis AA, Silvestros SS. Analysis of osteoblastic gene expression in the early human mesenchymal cell response to a chemically modified implant surface: an in vitro study. Clin Oral Implants Res. 2011;22(5):530-537. 2494 P.O. Box 1200, Shenyang 110004