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1 f zu{ } 19 í } Çf Chinese Journal of Tissue Engineering Research February 5, 2015 Vol.19, No.6 ÎÎÂµ«Œz o ƒ žq Í q k i n ˆ ¾( É Þ{ œ Ä Þ É ù ) s 1 ÎÎÂµ«Œz o ž oo² g ¼ } qu{ ÎÎÂµ«Œz o žq Í Œê qx ŒÉäg m ž~ y Íºüõ q 2 ÍŸ ÎÎÂµ«Œ ž ƒfùœéä ž~¼q Œ õ m ž~q µéîî Âµ«Œ g É ŒÉ ž~¼qmà ö n u º q Œz o q o ž ooo q Â gg ÎÎÂµ«Œz o ƒ ž oo ºÀ Œ z ƒ ž ÎÎÂµ«Œ ž~¼qmà É ž~ m Ù n Ž çž y gè ÎÎ Â«Œz ÙŒÁ oo «Ù n Ž çž y «ÙÈr( A48) ÎÎÂµ«Œz o žoo ± q º n Í rq ÎÎÂµ«Œz o SD Ùý žq Í ÙŒ gùœ ÙŒ Î ÙŒÍ Ä SD Ùý ž äqä SD ÙÎÎÂµ«Œ Ù žqúý r f d úý } mkr z ý Í Ùú r ÉfŸ ž~¼qmà o ä SRY o ä z ý z ú r ž~ ž~¼qmà o ä Â Œ Îp d (P < 0.01) z r Éff SRY o ä Â ŒÇjp ž~ É p f Ÿ µ z ÎÎÂµ«ŒŒ ² ž ²³m ž ~ Ù ž qž ÎÎÂµ«Œ g É Œ ³² Øž~¼qmÀ qé ˆ Ù ž ƒqž~ m k 1981 É ù 2009 É Þ{ x À Ä À Ôd ýú É Þ{ œ Ä Þ É ù doi: /j.issn [ f É ú:r394.2 h u:b ú: (2015) ÈÍ q k i n ˆ¾. ÎÎÂµ«Œz o ƒ žq Í[J].f zu{ (6): Mechanism of bone marrow mesenchymal stem cells transplantation in the treatment of limb ischemia Bai Chao, Yang Kun, Li Xin-xi, Wang Yang, Tian Ye, Luo Jun (Department of Thyroid Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi , Xinjiang Uygur Autonomous Region, China) Abstract BACKGROUND: Bone marrow stem cell transplantation has good achievements in the treatment of lower limb ischemic disease, but it mechanism of action is still unclear. OBJECTIVE: To explore the mechanism of bone marrow mesenchymal stem cell transplantation for treatment of limb ischemia in Sprague-Dawley rats. METHODS: The abdominal aorta, iliac arteries and lumbar arteries below the renal artery were ligated to prepare hind limb ischemia models in female Sprague-Dawley rats. Bone marrow mesenchymal stem cells from male Sprague-Dawley rats were amplified, purified and injected into the rectus femoris of the ischemic right hind limb in female rat models; in the control group, equal volume of normal saline was injected. After transplantation 2, 4, 6 weeks, hematoxylin-eosin staining of the right rectus femoris, immunohistochemical staining of vascular endothelial growth factor and SRY immunohistochemical staining were performed. RESULTS AND CONCLUSION: After operation 2, 4, 6 weeks, the capillary count and the number of cells positive for vascular endothelial growth factor in the transplantation group were significantly higher than those in the control group (P < 0.01). SRY immunohistochemical staining showed positive cells appeared at the capillary Bai Chao, Master, Attending physician, Department of Thyroid Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi , Xinjiang Uygur Autonomous Region, China Corresponding author: Luo Jun, M.D., Chief physician, Department of Thyroid Surgery, First Affiliated Hospital of Xinjiang Medical University, Urumqi , Xinjiang Uygur Autonomous Region, China Accepted: P.O. Box 10002, Shenyang

2 wall, which scattered in the muscle tissue in the transplantation group. These findings indicate that the local injection of bone marrow mesenchymal stem cells can improve the blood supply of ischemic hind limbs in Sprague-Dawley rats. Bone marrow mesenchymal stem cells as paracrine cells can prompt the neovascularization of ischemic limbs in rats by increasing the secretion of vascular endothelial growth factor. Subject headings: Bone Marrow; Mesenchymal Stem Cell Transplantation; Arterial Occlusive Diseases Funding: the Natural Science Foundation of Xinjiang Uygur Autonomous Region, No A48 Bai C, Yang K, Li XX, Wang Y, Tian Y, Luo J. Mechanism of bone marrow mesenchymal stem cells transplantation in the treatment of limb ischemia. Zhongguo Zuzhi Gongcheng Yanjiu. 2015;19(6): Introduction Ãs tˆm q Î m q ÙŒväÁ o ž Á Œ~ ö o ž ž~o } ž ooqöoi² é oi² Î rðço ² é ž ooq ö oµ o² ÙŒo ÙŒväz Ø ³ q ƒ ž o Ä [1-3] g o y ¼v ooq o o q ÙŒ ³ q o v k º p Ú uoo ² ž~ e Á Œ j q vä mž íöm 20d 90 xý t Çož~¼mq ³² uu{ Ùg Í o² y o ž ooqùÿ Kawamura} ƒ pîî CD34 + Œq qˆœéä ¼qx Œ ƒ¼œ Ÿ ž~q¼ÿå d ÎÎ «ŒŒÉ w Œ ˆž~œƒ~ ¼ ŸÅ¼qäq Œ ²ž~¼m g f Tateishi-Yuyama}Ë ³ nžƒîî Œz o ž oo Ëož~ m g nq f v2003 o}1 ŽƒÎÎ Œz o žooqo [4] Ãý ¹ ü Â o È [5-7] ÎÎÂµ«ŒnÎÎÉy º ýéä q Œ [8-10] ùxéäg ŒqŒ ³²tg ÎÎ ŒÉägž~¼q Œ ² žq mž~ ž qž o ž oo Žƒ Œz ² o ž ooq y [11-12] ŽƒÎÎ Œz Œ ² mž~ ž ƒž Ø³q oq ü oo Á Â Ÿö ÅŒ Éï g ž oo q o Èý o q rð pžƒ Œz ² ž mž~ q Í Ù É rºq Œ ²mÀ Ÿ² ² õ o Œ z r rð uq q Í ÍÍ SD Ùý ž oxîî Œz o mkr d r ± ä ÎÎÂµ«Œz o ƒ žqo ö Í u º q Œz o q o ž ooo q Â gg ÎÎÂµ«Œz o ƒ ž oo 1 Materials and methods Ã dùg Í Âö Íp Ž n çy Ùg Íf ÍÙg SPF ÁˆSD Ù50ù n nçy Ùg Íf mr ù ú SCXK( ) ˆn ù ú SCXK( ) ÍÙgˆ n xö Í çãk ~ ³² Â ú A ÙgÊ» %70% É³g m/s d Ø ü12 h ºf2ù4 ÚÄ SD Ùnp Œ» ƒ«146.5 g 48ùÄ SD Ùnp³ ÙÚ(5.0±0.6) ƒ«(156±0.6) g ÎÎÂµ«Œz osd Ù ƒ ž Í ng Ï y Ïöy ƒ DMEM» ƒzé g10% fž (g ) Ù ÙCD29 CD34 CD44 CD45ê Ã ƒ ÙSRY Ã ƒ Ùž~¼qmÀ Ã ƒ Leica DMI 4000B ³ À ç É Leica 2135Éf Leica HI1210 f HF240 CO 2»~ Jouan BR4y SW-CJ-2FDötêÅÂä ú Œy OPMI pico p À ˆ Gibco¹ú ˆ Hyclone¹ú ˆ Sigma¹ú ˆ Santa Cruz¹ú ë ë¹ú ë¹ú ë¹ú Ë mgçoy Â¹ú ¹ú Å { Â¹ú ˆ Becton-Dickinson¹ú Carl Zeiss Meditec AG ISSN CN /R CODEN: ZLKHAH 929

A C D 96.9% 3.6% 87.1% 0.1% 1 Œy ÎÎÂµ«ŒŸÅ Ÿ² Figure 1 Flow cytometry detection of bone marrow mesenchymal stem cell surface marker expression A x CD29 Ÿ²ig 96.9% B x CD34 Ÿ²ig 3.6% C x CD44 Ÿ²ig 87.

1% A B Ÿ 2 ü Ùú r ž~¼qmà o ä Â Œ (x _ ±s n=8) Table 2 Comparison of cells positive for vascular endothelial growth factor immunohistochemical staining of rat s right rectus femoris 4 z ý 6 z d SRY Â

right rectus femoris Ì z ý2 z ý4 z ý6 d 17.50±4.57 12.63±5.63 z 50.00±14.02 52.25±10.38 F P Ÿ z ý 2 4 6 z ú r ž~ p d (P < 0.

xý Ã3 dé 1 «Œ ²Í r80%90%»r ô» PBSÀ 2³ý n0.25%œ q ä «Œ Ä Œý d(68) 10 6 L -1 q Œ ²Ÿ ~x» ²n}5x Œ²Ÿz q Œn Œy ŸÅ Ÿ²Â i ( 1) CD29g96.9% CD44g87.1% CD34g3.6% 930 38.863 90.148 12.88±7.81 46.13±11.73 44.

3 A C D 96.9% 3.6% 87.1% 0.1% 1 Œy ÎÎÂµ«ŒŸÅ Ÿ² Figure 1 Flow cytometry detection of bone marrow mesenchymal stem cell surface marker expression A x CD29 Ÿ²ig 96.9% B x CD34 Ÿ²ig 3.6% C x CD44 Ÿ²ig 87.1% D x CD45 Ÿ²ig 0.1% A B Ÿ 2 ü Ùú r ž~¼qmà o ä Â Œ (x _ ±s n=8) Table 2 Comparison of cells positive for vascular endothelial growth factor immunohistochemical staining of rat s right rectus femoris 4 z ý 6 z d SRY Â ŒÉ À ( 400) Figure 4 Distribution of SRY positive cells at 6 weeks after cell transplantation ( 400) z (A)SRY Â Œ p d (B) dé Ÿ 1 ü Ùú r ž~ (x _ ±s, n=8) Table 1 The number of capillaries in rat s right rectus femoris Ì z ý2 z ý4 z ý6 d 17.50± ±5.63 z 50.00± ±10.38 F P Ÿ z ý z ú r ž~ p d (P < 0.01) Í ÎÎÂµ«Œ» n¹îî»»îî Âµ«Œ Âj f 2ù Ä SD Ù Î ŒÎ ÿ ÅÄ Ä ü100 U/mLqPBSf 15 min npbsà 2³ Ã Î n¾~ 5 ml»ƒ (ÿƒzé g10% fž 2 mmol/l L Œ Œ ÅÄ Ä ü100 U/mLqL-DMEM» ) ÎÎ ÀÇ Í ê Œ x(68) 10 9 L -1 Œ yp rf 48 hýë é xý Ã3 dé 1 «Œ ²Í r80%90%»r ô» PBSÀ 2³ý n0.25%œ q ä «Œ Ä Œý d(68) 10 6 L -1 q Œ ²Ÿ ~x» ²n}5x Œ²Ÿz q Œn Œy ŸÅ Ÿ²Â i ( 1) CD29g96.9% CD44g87.1% CD34g3.6% ± ± Ì z ý2 z ý4 z ý6 d 14.63± ±5.10 z 36.50± ±4.03 F P ± ± Ÿ z ý z ú r ž~¼qmà o ä Â Œ p d (P < 0.01) CD45g0.1% }üîîâµ«œg ÍÙgÉ 48ùÄ SD ÙÃ Ég d z 24ù ý ž Í ö ý k Ùn Œ 60 mg/kg Ø zì nœ µ n0.5%v} öœîð ² Åý ž µ féø À 3.0 cm ý ~ É ý Å gùœ ÙŒ Î ÙŒ ÙŒ n5-0d gùœ öœî ÙŒ n11-0d 24 Ù Œ º ÙØ ýíœê }2» ² 3 d ÅÄ ( U/ kg) ý5 d ÎÎÂµ«Œz p³ ý}5 Í Ùú r ² 5f ÎÎÂµ«Œ ù 0.25 ml ÿîîâµ«œ f d Ùr üµƒ 0.25 mlmkr g z ý2 4 6 f Â ÉÌ d z ü8ù Ù ú r Í Éf 10 min r j Éä ² 15 min j Œ pn ² { Ã ² À400 5f ž~ ž~¼qmà o ä f Œœ P.O. Box 10002, Shenyang

4 A B C D E F 2 ü Ùú r ( 400) Figure 2 The hematoxylin-eosin staining of rat s right rectus femoris ( 400) A B C ÉÌgz ýz Ùú r D E F ÉÌgz ý d Ùú r n ù z ž~ p d z 4 6 Œ pz 2 A B C D E F 3 ü Ùž~¼qmÀ o ä Œ ( 400) Figure 3 Number of cells positive for vascular endothelial growth factor (immunohistochemical staining, 400) A B C ÉÌgz ýz Ùú r D E F ÉÌgz ý d Ùú r n ù z ž~¼qmà o ä Â Œ p d ä ôã¼ ² äg ñ Å ož Á mg p DAB { Ã ² À400 5f ž~¼qmà o ä Â q Œ SRY o ä f Œœ ä ô Ã¼ ² äg ñ Å ož Á mg p DAB À SRY Â ŒqÉ À É nspss 17.0±z²Ÿ É «xx _ ±sÿx nê É Í P < 0.05g i ISSN CN /R CODEN: ZLKHAH 931

5 932 q }. ÎÎÂµ«Œz o ƒ žq Í 2 Results 2.1 ð 48ùSD ÙÃ Ég d z ü24ù f² Œ À 2.2 ê É Í z ý2 4 6 e Ùú r ž~ ± i(p < 0.01) Ÿ z ~p d Ÿ j Â ê É Í z ý2 4 6 ú r ž ~¼qmÀ o ä Â Œ e ± i(p < 0.01) z ~p d Ÿ SRY¾ ã z ý6 z ù µé ž~¼q Œ Â f Â qsry Â ŒÉ d SRY Œ 4 3 Discussion ÙŒo ³ q ƒ ž i±îqoo g o y ÈùŒˆµÉ qo Ã± v Ä Œz g o ÎÎ Œ rð ²nqy Œ [13] ù ƒ ž ž~ m ž Ø oo Î i [14-15] u{z Œq ¼m }ƒ¼± Í zîî Œz o ž ooq nz º i [16] ÍoxÎÎ Œz osd Ùý ž mkr q d r ± ä ÎÎÂµ«Œz o ƒ žqo ö Í u{öj Ù ž ƒz ÎÎÂµ«Œý f ž~ z ý2 ž~ ²ÍÎ Ÿ ÎÎÂµ«Œ žj fœ ˆ ž ²³m ž~ ž~¼qmà y ž~¼q Œ g Î q dé [17-18] ž~¼qmà qøœg ¼q Œü q Ø³ž~ ²ž~m [19-24] ž~³² [25] ž~ g ö ÎÎÂµ«Œz ýùx~û Í žµƒ [26-27] ùxˆ ž ƒ fqž~¼qmà Ø [28-31] Øqž~¼qmÀ kùx ØÎÎ Âµ«Œq [32-34] u{z ž~¼qmà Â Œ ² p d Ÿ ÎÎÂµ«Œz ùx Ø ž ¼ž~¼qmÀ qÿ ² Œv Øqž ~¼qmÀ q ùœ ÎÎÂµ«Œ g É Œ rm [35] SRY Y ƒ À mgä Ìq f SRY Â q Œí pä SD ÙÎÎÂµ«Œ z µé ž~¼q ŒSRY Â Â qsry Â Œ Ÿ ÎÎÂ µ«œ ž ƒfùœéä ž~¼q Œ õ m ž~q «h Â kcýú kcì kcýú ò À kc c À d kn ýú k d ÌrÀ{ À À í q d k «n t 2009 wethical issues in animal experimentationx üì íd SRY¾ À Ú ¾ Y Ú ¾ Šd SRY¾ Yp11.3 ù} j 1.1 kb þ 204 ¾ ~ d u k À í À d 4 õ h References [1] Alavi A, Sibbald RG, Mayer D, et al. Diabetic foot ulcers: Part I. Pathophysiology and prevention.j Am Acad Dermatol. 2014; 70(1):1.e1-18; quiz [2] Schaper NC, Andros G, Apelqvist J, et al. Diagnosis and treatment of peripheral arterial disease in diabetic patients with a foot ulcer. A progress report of the International Working Group on the Diabetic Foot.Diabetes Metab Res Rev. 2012;28 Suppl 1: [3] O'Loughlin A, McIntosh C, Dinneen SF, et al. Review paper: basic concepts to novel therapies: a review of the diabetic foot.int J Low Extrem Wounds. 2010;9(2): [4],µ²l,,}.ŽƒÎÎ Œz oe ž 1 [J].f n y,2003,23(11):670. [5] Â ì, e, r,}. ü z ² qžƒ Œ o o ž ž~o qã du{[j].fég ç :n f,2013,7(14): [6] é,â,àsé,}.žƒ ž Œz o o ž [J].f üg,2011,27(9): [7] š,êà,â,}.žƒîîêf Œz o Ù ŒÁ o[j].f zu{,2013,17(10): [8] Hatzistergos KE, Quevedo H, Oskouei BN, et al. Bone marrow mesenchymal stem cells stimulate cardiac stem cell proliferation and differentiation.circ Res. 2010;107(7): [9] Kassis I, Vaknin-Dembinsky A, Karussis D. Bone marrow mesenchymal stem cells: agents of immunomodulation and neuroprotection.curr Stem Cell Res Ther. 2011;6(1): [10] Mohamadnejad M, Sohail MA, Watanabe A, et al. Adenosine inhibits chemotaxis and induces hepatocyte-specific genes in bone marrow mesenchymal stem cells.hepatology. 2010; 1(3): [11] Dhinsa BS, Adesida AB.Current clinical therapies for cartilage repair, their limitation and the role of stem cells.curr Stem Cell Res Ther. 2012;7(2): P.O. Box 10002, Shenyang

6 [12] Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet. 2002; 60(9331): [13] Bai K, Huang Y, Jia X, et al. Endothelium oriented differentiation of bone marrow mesenchymal stem cells under chemical and mechanical stimulations.j Biomech. 2010;43(6): [14] Schiavetta A, Maione C, Botti C, et al. A phase II trial of autologous transplantation of bone marrow stem cells for critical limb ischemia: results of the Naples and Pietra Ligure Evaluation of Stem Cells study.stem Cells Transl Med. 2012; 1(7): [15] Hamano K, Li TS, Kobayashi T, et al.the induction of angiogenesis by the implantation of autologous bone marrow cells: a novel and simple therapeutic method.surgery. 2001; 130(1): [16] Park JS, Hashi C, Li S.Culture of bone marrow mesenchymal stem cells on engineered matrix.methods Mol Biol. 2010;621: [17] Bonnefond A, Saulnier PJ, Stathopoulou MG, et al. What is the contribution of two genetic variants regulating VEGF levels to type 2 diabetes risk and to microvascular complications. PLoS One. 2013;8(2):e [18] Liu Y,Zhu SQ,Wang YF,et al.neuroprotective effect of ischemic preconditioning in focal cerebral infarction: relationship with upregulation of vascular endothelial growth factor. Neural Regen Res. 2014;9 (11): [19] Liang K, Jiang Z, Zhao B, et al.the expression of vascular endothelial growth factor in mast cells promotes the neovascularisation of human pterygia.br J Ophthalmol. 2012;96(9): [20] Odemis V, Boosmann K, Heinen A, et al. CXCR7 is an active component of SDF-1 signalling in astrocytes and Schwann cells. J Cell Sci. 2010;123(Pt 7): [21] Î, ², Æ,}. ž~¼qmà ÊÎÎÂµ«Œz Ù:Îï È ² o ž~m [J]. f zu{,2012,16(27): [22] Ê¾. ÎÎÂµ«Œ É ž~m qu{² [J]. çy,2011,17(1): ,139. [23], Ë,,}. VEGF Ê ÎÎÂµ«Œq u{[j].f zu{ g,2008,12(12): [24] Î,, Ì,}. ž~¼qmà ± ÎÎÂµ«Œz ² ž ž~m qu{[j].f n¼y,2006,26(20): [25] Kwon MJ, An S, Choi S, et al. Effective healing of diabetic skin wounds by using nonviral gene therapy based on minicircle vascular endothelial growth factor DNA and a cationic dendrimer.j Gene Med. 2012;14(4): [26] Xiong N, Zhang Z, Huang J, et al.vegf-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for Parkinson's disease.gene Ther. 2011;18(4): [27] Hollweck T, Marschmann M, Hartmann I, et al. Comparative analysis of adherence, viability, proliferation and morphology of umbilical cord tissue-derived mesenchymal stem cells seeded on different titanium-coated expanded polytetrafluoroethylene scaffolds. Biomed Mater. 2010;5(6): [28] Fan W, Crawford R, Xiao Y.The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization.differentiation. 2011;81(3): [29] Ÿ, º,,}. ÎÎÂµ«ŒŠüž~¼qmÀ o ƒ žq Íu{[J].fé ³ y, 2012, 27(1): [30] j,â ~,ih,}. ÎÎÂµ«Œz ž qž ~ mö -Ãrq [J]. uç g,2012,32(4): [31] ¾, Ü, ˆü,}. { ÎÎÂµ«Œ ý ž Ùž q [J].f ¾,2009,29(12): [32] Deng J, Zou ZM, Zhou TL, et al. Bone marrow mesenchymal stem cells can be mobilized into peripheral blood by G-CSF in vivo and integrate into traumatically injured cerebral tissue. Neurol Sci. 2011;32(4): [33] Tang JM, Wang JN, Zhang L,et al.vegf/sdf-1 promotes cardiac stem cell mobilization and myocardial repair in the infarcted heart.cardiovasc Res. 2011;91(3): [34] Thibeault S, Rautureau Y, Oubaha M, et al. S-nitrosylation of beta-catenin by enos-derived NO promotes VEGF-induced endothelial cell permeability. Mol Cell. 2010;39(3): [35] Guiducci S, Manetti M, Romano E, et al. Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis exhibit a paracrine machinery and stimulate angiogenesis in vitro.ann Rheum Dis. 2011;70(11): ISSN CN /R CODEN: ZLKHAH 933

Microsoft Word - JA 旅運經營學-講義-01.doc

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