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1 : plasmid VE GF, *,, * * * * * * * * * =A b s t r a c t = Ch ar ac t e ri s tic s of g e n e tra n s f e r an d V E GF e x p re s s i on u s in g n ak e d p la s m i d v e c t or s in th e rat h e art Jin - Ok Jeong, M.D. *, Sun - Jin P ark, B.S. *, Jeong - Eun Huh, M.S. *, Eun - Ah Jung, M.S.*, Ju Hyeon Oh, M.D. *, Hyeon - Cheol Gwon, M.D. *, YoungJoo Lee, Ph.D., Sunyoung Kim, Ph.D., Jeong - Euy Park, M.D. *, Won Ro Lee, M.D. *, Eun - Seok Jeon, M.D. and Duk - Kyung Kim, M.D. * Department of M edicine, Sungkyunkwan University School of M edicine, Cardiac and Vascular Center, Samsung M edical Center *, Samsung B iomedical R esearch Institute Institute of M olecular and Genetic B iology, Seoul N ational University Division of Cardiology, Department of M edicine, Chungnam N ational University B ackg round : T he purpose of this study was to compare gene expression among newly designed eukaryotic expression vectors, and to characterize the pattern of vascular endothelial growth factor (VEGF ) expression using the most potent plasmids DNA vector. Meth ods : After exposure of a beating rat heart (Sprague-Dawley, g), 5 different types of plasmid DNA was injected directly into the myocardium. Reporter protein was analyzed by ELISA in the extracted heart. Re s ult s : T he vector harboring cytomegalovirus (CMV) promoter and enhancer induced the strongest expression of reporter gene (chloramphenicol acetyl transferase; CAT ) compared to those of pc3.1, pef 1a, RSV, pactin in the rat heart via direct injection of plasmid DNA into the apex (p <0.001). Using pcn- CAT, gene expression showed a dose- dependent response over a range of CAT expression could be detected up to 30 days after 10 of pcn- CAT injection with the maximal expression on day 5. In X- gal staining of injected pcn- lacz gene, - galactosidase was found only around the needle track in the apex. The expressed hvegf 12 1 had biologic activity with vascular permeability assay (Miles assay) in guinea pigs. After injection of pcn-hvegf 12 1 into the apex of the rat heart, the expression of VEGF protein was dose- dependent over the range of 25 and : : :, 50,,, ( ) E -mail : - 3 -

2 Korean Journal of Medicine : Vol. 60, No. 1, VEGF expression was detected up to 14 days with its peak on day 2 after injection of 250 of pcn- hvegf When plasmid was injected into the apex of the rat heart, the expression of VEGF in the heart showed concentration gradient from the apex to the base. However, the expression of CAT was detected only in the apex. Conclu s ion : Plasmids vector with hcmv IE promoter/ enhancer will provide clear advantages over other previously developed plasmids and the information regarding the behaviors of VEGF expression may be useful in angiogenic gene therapy of the heart.(korean J Med 60:3-15, 2001) Key W ords : Gene therapy; Plasmids; Endothelial growth factors; Promoter regions(genetics) ( ) (angiogenic gene therapy) (angiogenic factor)., 1-4 )... plasmid (prom oter ),, housekeeping, - (tissue specific). intron 1 enhancer cis- acting element 5, 6 ). intron plasmid,, reporter gene chloramphenicol acetyl transferase (CAT ) hvegf Plasmid pcdna3.1- CAT, pcn- CAT, pcn- lacz, pef - CAT, pact- CAT, prsv- CAT Promega. pcn, pef, pact pcdna3.1 backbone pcdna bp CMV IE Invitrogen. Figure 1 7). pcdna3.1 pcn cytomegalovirus immediate early (CMV IE), prsv rous sarcome virus, pef pact housekeeping gene elongation factor 1- alpha beta actin. pcn pcdna3.1 CMV exon 1, intron 1, exon 2 AT G. pef pact exon 1, intron 1, exon gram Sprague- Dawley rat. Ketamine ( 50 mg/ ml) Xylazine ( mg/ ml) 3 : ml. (Natume, Japan, model PT ) midline - 4 -

3 Jin- Ok Jeong, et al : Characteristics of gene transfer and VEGF expression using naked plasmid vectors in the rat heart. DNA purification kit (Qiagen ) plasmid DNA 50 L 29 gage 1 ml needle 2 mm black silk. Cephalosporine sodium (Cefamezin, 1.0 g/ vial) 0.1 g/ 1 ml normal saline, cage.. CAT ELISA, VEGF ELISA, X- gal. 3. CAT ELISA ice- cold PBS 1/ 3 2 ml CAT lysis buffer (Boehringer Mannheim ) homogenize 4 o C, 12,000 rpm 10. 1/ L Braford (Bio- Rad ) CAT ELISA kit (Boehringer Mannheim ) CAT. 4. LacZ ice- cold PBS 1/ 3 tissue mold O.C.T (Lipshow ) mold - 70 o C. 10 ice- cold 4% paraformaldehyde (ph 7.4, Sigma ) 5. 2 mm MgCl2/ PBS (ph 7.4, Sigma ) X- gal (4 mm potassium ferricyanide, 4 mm potassium ferrocyanide, 2 mm MgCl2, 400 / ml X- Gal) 4 lacz eosin 80% alcohol, 90% alcohol, 100% alcohol, xylene cover glass. (Olymphus ) 40, ACP-hVEGF12 1 VEGF adeno- associated virus IT R (inverted terminal repeat) ACP EcoRI site PCR cloning human VEGF 12 1 cdna ACP- hvegf Transient transfection VEGF ELISA 293T 10% fetal bovine serum (FBS)/ DMEM (Gibco BRL ) 6 well plate 70% confluent. T ransfection 3 ACP- hvegf 12 1 DNA/ 200 L serum free media (OPTI- MEM, Gibco BRL ) liposome FuGENE 6 (Roche ) 9 L % FBS/ DMEM 48 VEGF ELISA kit (R&D ). 7. Miles assay Vascular permeability factor VEGF Miles assay. Male guinea pig ( g) (, ). Evans blue dye PBS 0.5% (w/v), 0.22 filter 1 ml (femoral vein). 30 ACP ACP- hvegf 12 1 transfection 293T 1 ml 100 L (intradermal) evans blue dye. 8. VEGF ELISA CAT ELISA homogenize 4 o C, 12,000 rpm

4 : / L Braford (Bio- Rad ). 200 L (calibrator 1:1 ) VEGF. 10. InStat program (version 2.02) Kruskal- Wallis test, p 0.05,. 1. 1) CAT 56. pcdna3.1- CAT, pcn- CAT 15, prsv- CAT, pef- CAT 9, pact- CAT 8 plasmid DNA CAT ELISA. CAT pcn- CAT CAT pg/ mg, pcdna3.1- CAT pg/ mg, prsv- CAT pg/ mg, pef- CAT pg/ mg, pact- CAT pg/ mg CAT (Figure 2). pcn CAT pcdna3.1, prsv, pact (p <0.001), pef (p <0.01). pcdna3.1 prsv, pef, pact CAT. CMV intron 1 pcn- CAT pcdna3.1- CAT ) pcn-cat dose, time-response curve pcn- CAT DNA. 3 CAT ELISA. pcn- CAT plateau, (Figure 3A). Fig ure 1. Structure of plasmid vectors. Arrows indicate direction of transcription. pcdna3.1 contains hcmv promoter (Invitrogen Inc.). pcn contains hcmv promoter, exon 1, intron 1 and partial exon 2. prsv contains RSV promoter. pef contains elongation factor 1- promoter, exon 1, intron 1 and partial exon 2. pact contains beta actin promoter, exon 1, intron 1 and partial exon 2. hcmv, human cytomegalovirus; RSV, rous sarcoma virus Fig ure 2. Comparison of chloroampenicol acetyltransferase expression level among different promoters of plasmid DNA in the rat heart. Each 30 pdna was directly injected into the apex with pcdna3.1 (n=15), pcn (n=15), prsv (n=9), pef (n=9) and pact (n=8). CAT assay was done by ELISA with extracted heart at 3 days after plasmid injection. The level of CAT gene expression using pcn was significantly higher than those of other promoters (* p <0.001). Values are mean S.E.M

3 to 10, then plateaued, followed by decrease of gene expression in higher doses. Values are mean S.E.M. B. Time-response of the injected pcn-cat expression in the rat heart (3 rats for each group).

5 11 : plasmid VEGF Fig ure 3. A. Dose- response of the injected pcn- CAT expression in the rat heart (3 rats for each group). CAT assay was done by ELISA with extracted heart in 3 days after direct injection of pdna into the rat heart. There was a dose- responsive relation from 0.3 to 10, then plateaued, followed by decrease of gene expression in higher doses. Values are mean S.E.M. B. Time-response of the injected pcn-cat expression in the rat heart (3 rats for each group). CAT assay was done by ELISA with extracted heart after direct injection of 10 of pcn- CAT. CAT expression was detected at 1, 3, 5, 7, 14, 30 days after injection with maximal expression at 5th day. Values are mean S.E.M. F ig ure 4. Histochemical analysis of - galactosidase expression in the rat heart, five days after direct injection of 10 of pcn- lacz DNA. Dark blue staining indicates active expression of - galactosidase. A. Expression of pcn- lacz: - galactosidase could be detected around the needle track in the apex (original magnification 40). B. Expression of pcn- lacz and infiltration of inflammatory cells (original magnification 200). C. Expression of pcn- lacz purified with endotoxin- free column: - galactosidase was expressed and the degree of infiltration of inflammatory cells was similar to the one with plasmids without purification of endotoxin (original magnification Cytoplasmic 200). lacz, pcn- CAT DNA 10. CAT 5 30 (Figure 3B). 3) LacZ pcn- lacz DNA 10, 5 X- gal (needle injection) (Figure 4A). - galactosidase (Figure 4B). E- coli endotoxin contamination, endotoxinfree column DNA. Endotoxin- free column - galactosidase

Korean Journal of Medicine : Vol. 60, No. 1, 2001 (Figure 4C). 2. VEGF 1) ACP-hVEGF12 1 VEGF ACP- hvegf 121 VEGF 293T ACP- hvegf 12 1 FuGENE 6 transient transfection, VEGF ELISA.

3) pcn-hvegf12 1 dose, time-response curve 2) hvegf12 1 (Miles permeability assay) ACP- hvegf 121 VEGF guinea pig evans blue dye guinea pig.

6 Korean Journal of Medicine : Vol. 60, No. 1, 2001 (Figure 4C). 2. VEGF 1) ACP-hVEGF12 1 VEGF ACP- hvegf 121 VEGF 293T ACP- hvegf 12 1 FuGENE 6 transient transfection, VEGF ELISA. Negative control (ACP transfection) ACP- hvegf 12 1 transfection VEGF ( ng/ ml versus ng/ ml p <0.001). (Figure 5). 3) pcn-hvegf12 1 dose, time-response curve 2) hvegf12 1 (Miles permeability assay) ACP- hvegf 121 VEGF guinea pig evans blue dye guinea pig. VEGF, hvegf 12 1 transfection permeability hvegf 12 1 VEGF, ACP- mvegf 120 Fig ure 5. Miles assay of VEGF. Biologic activity of VEGF 12 1 w as demonstrated with vascular permeability assay in guinea pig. Intravenous injection of Evans blue dye via femoral vein followed by intradermal injection of standard proteins or supernatant of ACP - hvegf 12 1 tansfected 293T cells. T here were blue dots around intradermal injection site showed extravasation of evans blue dye due to increased vascular permeability of VEGF. Left lane was recombinant murine VEGF peptide as standard (ST D). Dot size and intentsity of deep blue color were VEGF dose- dependent. Right lanes showed larger deep blue dots around intradermal injection of ACP - hvegf 12 1 and ACP - mvegf 12 0 conditioned media than ACP conditioned media as a negative control. Fig ure 6. A. Dose- response of pcn- hvegf 12 1 expression. Each pdna was directly injected in the rat heart, where n=3 for each group. VEGF ELISA assay was done 2 days after injection. Expression of VEGF was dose- dependent from 25 to 500 of pcn- hvegf 12 1 DNA injection. Values are mean S.E.M. B. Time- response of pcn- hvegf 12 1 expression after injection of 250 pcn-hvegf12 1 in the rat heart, where n=3 for each group. VEGF expression could be detected from 1 days upto 14 days after injection with maximal expression on day 2. Values are mean S.E.M

7 Jin- Ok Jeong, et al : Characteristics of gene transfer and VEGF expression using naked plasmid vectors in the rat heart ACP- hvegf 121 hvegf 121 c DNA pcn pcn- hvegf VEGF ELISA. VEGF DNA (Figure 6A). pcn- hvegf VEGF 2 14 VEGF (Figure 6B). 4) pcn-hvegf12 1 VEGF VEGF 10 pg/ mg pcn CAT 1,800 pg/ mg. VEGF 1, VEGF 12 1 heparin- binding domain, VEGF Fig ure 7. Spatial distribution of protein produced by gene injection. A. Spatial distribution of CAT protein after a single injection of pcn- CAT 10 in the apex of the rat heart. T hree hearts at day 3 after injection were analyzed. CAT protein was detected only in the apex. Values are mean S.E.M. B. Spatial distribution of VEGF protein after a single injection of pcn- hvegf in the apex of the rat heart. Six hearts at second day after injection were analyzed. Concentration gradient of VEGF protein was noted from the apex to the base. Values are mean S.E.M.. plasmid DNA VEGF CAT, pcn- hvegf (n=6) pcn- CAT 10 (n=3). VEGF 2, CAT 3 4 VEGF, CAT. CAT pg/ mg, pg/ mg, pg/ mg, pg/ mg plasmid DNA (Figure 7A). VEGF pg/mg, pg/ mg, pg/ mg, pg/mg VEGF plasmid DNA VEGF (Figure 7B). 1.. retrovirus, adenovirus, adenoassociated virus, retrovirus,. in vivo 1% 8-10 ), (granulation tissue) 14% 11). Adenovirus, adenovirus plasmid DNA , 13 ). adenovirus 100% lacz 14), adenovirus rabbit, plasmid DNA 50 15). adenovirus - 9 -

8 : adenovirus immunogenicity, 2 13). ornithine transcarbamylase adenovirus adenovirus. adenovirus negative control, adenovirus. adenovirus. Adeno- associated virus,,,,. 2. naked DNA naked DNA. Naked DNA,,,,. naked DNA., naked DNA (striated muscle). Naked DNA 8 16 ), 50 17, 18 )., naked DNA 19 )., (hypoxia) upregulation 20 )., retovirus VEGF (angioma- like lesion) 2 1),. w all tension naked DNA. Naked DNA naked DNA. naked DNA 1-3 ).. plasmid, DNA,,. 3. Naked DNA naked DNA in vivo, reporter gene CAT pcn > > pef > pcdna3.1 > prsv > pact pcn CAT. pcdna3.1 Invitrogen

9 11 : plasmid VEGF,, Isner. pcn pcdna Buttrick 22 ) adult female Wistar rats 100 naked DNA luciferase reporter. RSV, - myosin heavy chain ( MHC) 1- antitrypsin RSV >> MHC > 1- antitrypsin, RSV MHC 20. RSV. pcn, pcdna3.1, prsv, pef, pact. CMV IE Overell 23 ) primary B lympocytes CMV- IE Moloney murine leukemia virus long terminal repeat (MoMLV LT R) SV40 early region reporter gene CAT, Hippenmeyer 24 ) quail cell SV40 early, mouse metallothionein I, CMV- IE CMV. pcdna3.1 prsv CAT. CMV RSV. EF - 1 human EF - 1. intron 1 Sp1, Ap1. pef intron 1 Nagata pef321 5, 6). pef CMV. Beta actin housekeeping gene. Beta actin,. pcn CMV IE exon 1, intron 1, exon 2. CMV EF 1- AT G site 5' - untranslated region. Simari 25 ) human CMV IE CMV 5' - untranslated sequence exon 1 intron 1 5.7, RSV 10.3 (enahncer). pcn pcdna CMV- IE / (, exon 1, intron 1 ). 4. naked DNA CAT dose, time-response pcn- CAT plateau,. Li 26) CD1 RSV rat - MHC naked plasmid DNA luciferase activity, RSVluc plateau. CAT

10 Korean Journal of Medicine : Vol. 60, No. 1, Heller 27 ) electroporation luciferase - galactosidase naked DNA 25, , Harsdorf 28) mouse sarcoma virus- CAT CAT , reporter gene. pcn- hvegf 12 1 VEGF 500,. pcn- CAT 10 5, 30. Li 26) CD1 RSV rat - MHC naked plasmid DNA luciferase activity luciferase 1, Lin 18) in vivo 6-11 Sprague- Dawley RSV galactosidase plasmid DNA 4. Buttrick 22 ) prsv- CAT prsv- Luc naked DNA 100 Wistar rat CAT luciferase, %, %, %, pcn- CAT - naked DNA. 5.. Li 26 ) naked DNA 1-2 mm, luciferase, (septum), c- myc myc- specific monoclonal antibody c- myc. Buttrick 22 ) Wister rat prsvluc mm. Lin 18 ) prsv - galactosidase Sprague- Dawley rat - galactosidase. - galactosidase, naked DNA naked DNA., Quiagen DNA purification E. coli endotoxin, endotoxin- free column DNA endotoxin., E- coli - galactosidase, lacz. - galactosidase. VEGF 1), VEGF naked DNA. 6. CAT VEGF pcn- hvegf 121 naked plasmid VEGF pg/ mg pcn- CAT CAT pg/ mg, time- response VEGF

11 Jin- Ok Jeong, et al : Characteristics of gene transfer and VEGF expression using naked plasmid vectors in the rat heart 2 CAT 5. CAT,., VEGF 1, VEGF 12 1 heparin- binding domain, VEGF. pcn- lacz LacZ, pcn- CAT CAT plasmid DNA VEGF VEGF. hvegf 121. pcn mrna level pcn- CAT pcn-hvegf 121 VEGF time- response, RT - PCR mrna naked DNA. CAT 10, 5 30., hvegf 12 1 cdna VEGF vascular permeability., pcn- hvegf 12 1 VEGF plasmid 500, 2, 14., VEGF plasmid DNA., CAT plasmid DNA. : naked DNA CMV intron 1 pcn. VEGF plasmid,, 14 VEGF kinetics VEGF. hvegf 12 1 cdna. : intron plasmid,, reporter gene chloramphenicol acetyl transferase (CAT ) hvegf : gram Sprague- Dawley rat plasmid DNA. :, hcmv IE intron 1 pcn., pcn- CAT reporter gene 1999 (HMP- 99-B ) (C98-002). R E F E R E N C E S 1) Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, Isner JM. Constitutive exp ression of phve GF1 65 af ter intramuscular gene transf er p romotes collateral vessel developm ent in patients with critical limb ischemia. Circulation 97: , ) Losordo DW, Vale PR, Symes JF, Dunnington CH, Esakof DD, Maysky M, Ashare AB, Lathi K, Isner JM. Gene therapy for myocardial angiogenesis:

12 : initial clinical results with direct myocardial inj ection of phvegf165 as sole therapy for myocardial ischemia. Circulation 98: , ) Symes JF, Losordo DW, Vale PR, Lathi KG, Esakof DD, Mayskiy M, Isne JM. Gene therapy with vascular endothelial growth factor f or inop erable coronary artery disease. A nn Thorac Surg 68: , ) Rosengart T K, Lee LY, Patel SR, Sanborn TA, Parikh M, Bergman GW, Hachamovitch R, Szulc M, Kligfield PD, Okin PM, Hahn RT, Devereux RB, Post MR, Hackett NR, Foster T, Grasso T M, Lesser ML, Isom OW, Crystal RG. A ngiogenesis gene therapy: phase I assessment of direct intramyocardial administration of an adenovirus vector exp ressing VE GF12 1 cdna to individuals with clinically significant severe coronary artery disease. Circulation 100: , ) Mizushima S, Nagata S. pef - B OS, a p owerful mammalian exp ression vector. N ucleic A cids R es 18:5322, ) Wakabayashi- Ito N, Nagata S. Characterization of the regulatory elements in the p romoter of the human elongation factor-1 alpha gene. J B iol Chem 269: , ) Lee YJ, Park EJ, Yu SS, Kim DK, Kim S. Imp roved exp ression of vascular endothelial growth factor by naked DNA in mouse skeletal muscles: imp lication for gene therapy of ischemic disease. B iochem B iophys R es Commun 272: , ) Ferry N, Duplessis O, Houssin D, Danos O, Heard JM. R etroviral-mediated gene transfer into hepatocytes in vivo. P roc N atl A cad Sci USA 88: , ) Gojo S, Kitamura S, Germeraad WT, Yoshida Y, Niwaya K, Kawachi K. Ex vivo gene transf er into myocardium using rep lication- defective retrovirus. Cell T ransp lant 5:S81-84, ) Huh JE, Kim DK, Byun JH, Park SJ, Jeon ES, Choe YH, Jung EA, Gwon HC, Park SW, Kim JS, Lee SH, Hong KP, Park JE, Cosset FL, Sei JD, Lee WR. Gene transf er into cultured cardiac myocytes m ediated by retrovirus. K orean Circ J 29: , ) Byun J, Huh JE, Park SJ, Jang JE, Suh YL, Lee JS, Gwon HC, Lee WR, Cosset FL, Kim DK. M yocardioal inj ury- induced fibroblast p rolif eration facilitates retroviral- mediated gene transf er to the rat heart in vivo. J Gene M ed 2:1-9, ) Kass-Eisler A, Falck-Pedersen E, Alvira M, Rivera J, Buttrick PM, Wittenberg BA, Cipriani L, Leinwand LA. Quantitative determ ination of adenovirusmediated gene delivery to rat cardiac myocytes in vitro and in vivo. P roc N atl A cad Sci USA 90: , ) Guzman RJ, Lemarchand P, Crystal RG, Epstein SE, Finkel T. Efficient gene transfer into myocardium by direct inj ection of adenovirus vectors. Circ R es 173: , ) Donahue JK, Kikkawa K, Johns DC, Marban E, Lawrence JH. Ultrarap id, highly eff icient viral gene transf er to the heart. P roc N atl A cad Sci USA 94: , ) Barr E, Carroll J, Kalynych AM, Tripathy SK, Kozarsky K, Wilson JM, Leiden JM. Efficient cathetermediated gene transf er into the heart using rep lication- def ective adenovirus. Gene Ther 1:51-58, ) Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL. Direct gene transf er into mouse muscle in vivo. Science 247: , ) Kitsis RN, Buttrick PM, McNally EM, Kaplan ML, Leinwand LA. H ormonal modulation of a gene inj ected into rat heart in vivo. P roc N atl A cad Sci USA 88: , ) Lin H, Parmacek MS, Morle G, Bolling S, Leiden JM. Exp ression of recombinant genes in myocardium in vivo af ter direct inj ection of DNA. Circulation 82: , ) Levy AP, Levy NS, Loscalzo J, Calderone A, T akahashi N, Yeo KT, Koren G, Colucci WS, Goldberg MA. R egulation of vascular endothelial growth factor in cardiac myocytes. Circ R es 76: , ) Banai S, Shweiki D, Pinson A, Chandra M, Lazarovici G, Keshet E. Up regulation of vascular endothelial growth factor exp ression induced by myocardial ischemia: imp lications f or coronary angiogenesis. Cardiovasc R es 28: , ) Springer ML, Chen AS, Kraft PE, Bednarski M, Blau HM. VE GF gene delivery to muscle: potential role f or vasculogenesis in adults. M ol Cell 2: , ) Buttrick PM, Kass A, Kitsis RN, Kaplan ML, Leinwand LA. B ehavior of genes directly inj ected into the rat heart in vivo. Circ R es 70: , ) Overell RW, Weisser KE, Hess BW, Ziegler SF, Pleiman CM, Maliszewski C, Grabstein KH. Eff icient gene transf er and exp ression in p rimary B lymphocytes. J Imm unol M ethods 141:53-62, ) Hippenmeyer PJ, Krivi GG. Gene exp ression f rom heterologous p romoters in a rep lication- def ective avian retrovirus vector in quail cells. P oult Sci 70: , ) Simari RD, Yang ZY, Ling X, Stephan D, Perkins

13 11 : plasmid VEGF ND, Nabel GJ, Nabel EG. R equirem ents f or enhanced transgene exp ression by untranslated sequences from the human cytom egalovirus immediate- early gene. M ol M ed 4: , ) Li K, Welikson RE, Vikstrom KL, Leinwand LA. Direct gene transf er into the mouse heart. J M ol Cell Cardiol 29: , ) Heller R, Jaroszeski M, Atkin A, Moradpour D, Gilbert R, Wands J, Nicolau C. In vivo gene electroinj ection and exp ression in rat liver. FEB S L ett 389: , ) von Harsdorf R, Schott RJ, Shen YT, Vatner SF, Mahdavi V, Nadal- Ginard B. Gene inj ection into canine myocardium as a usef ul model f or studying gene exp ression in the heart of large mammals. Circ R es 72: ,

Lumbar spine

Lumbar spine CT 32 111 DOI : 10.3831/KPI.2010.13.2.111 Lumbar Spine CT 32 Received : 10. 05. 23 Revised : 10. 06. 04 Accepted : 10. 06. 11 Key Words: Disc herniation, CT scan, Clinical analysis The Clinical

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歯60-1.PDF