서론 978 Korean Circulation J 1998;286:

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1 Original Articles Korean Circulation J 1998;286: 쥐경동맥손상모델에서 Herpes Simplex Virus Thymidine Kinase 유전자치료의협착방지효과 김동운 1 김영규 2 오태근 1 조명찬 1 김승택 1 Retrovirus-Mediated Herpes Simplex Virus Thymidine Kinase Gene Therapy for the Prevention of Stenosis in Rat Carotid Artery Injury Model Dong-Woon Kim, MD 1, Young Gyu Kim, MD 2, Tae Geun Oh, MD 1, Myeong-Chan Cho, MD 1 and Seung Taik Kim, MD 1 1 Department of Internal Medicine and 2 Neurosurgery, College of Medicine, Chungbuk National University, Cheongju, Korea ABSTRACT BackgroundHerpes simplex virus thymidine kinase HSVtk phosphorylates the prodrug ganciclovir to a nucleoside analog that inhibits DNA synthesis, causing cell death. Neighbouring nontransfected cells may be affected through a bystander effect, thereby amplifying the antiproliferative actions. This study was carried out to determine whether retrovirus-mediated HSVtk gene therapy could reduce intimal hyperplasia and prevent stenosis following balloon injury of the rat carotid artery. MethodsA replication-defective recombinant retroviral vector containing HSVtk cdna LtkSN was constructed. Cultured primary rat smooth muscle cells SMCs infected with this vector SMC / LtkSN were transplanted to the balloon injured rat right carotid artery. One week after transplantation, HSVtk gene therapy group was administered a 2-week treatment of ganciclovir 30 mg / kg / d. Three weeks after balloon injury and SMC/LtkSN transplantation, carotid arteriography was performed and carotid arteries were perfusion-fixed for histologic examination. ResultsCarotid arteriographic evaluation comparing with the uninjured left carotid artery showed that the mean luminal diameter of HSVtk gene therapy group n5, 853% was significantly larger than that of balloon injury only group n5, 655%. The neointimal mass of HSVtk gene therapy group was less than that of balloon injury only group. SMC / LtkSN transplantation without ganciclovir treatment group n3 showed asymmetric intimal proliferation probably because of gravitational pooling of seeding. There were inflammatory cell infiltrations at the gravity dependent portion of HSVtk gene therapy group. Conclusion Retrovirus-mediated HSVtk gene therapy following balloon injury of the rat carotid artery reduced neointimal expansion and arteriographic stenosis. Korean Circulation J 1998;286: KEY WORDSGene therapy Restenosis HSVtk gene. 977

2 서론 978 Korean Circulation J 1998;286:

3 방법 LtkSN의제작 979

4 재조합 retrovirus의제작 980 쥐혈관평활근세포배양및 Retroviral vector 감염 HSVtk 유전자치료모델 Korean Circulation J 1998;286:

5 유전자이식검정 981

6 실험동물의분류 통계적분석 982 결과 조직학적소견 경동맥조영술 Korean Circulation J 1998;286:

(A) Uninjured carotid artery (B) Balloon-injured carotid

cells (SMCs) expressing herpes simplex virus thymidine

Arterial segment seeded with SMCs expressing HSVtk with

7 A B C D A B C Fig. 1. Histological sections from representative arterial segments (40 magnification). All specimens were obtained 21 days after balloon injury. (A) Uninjured carotid artery (B) Balloon-injured carotid artery (C) Arterial segment seeded with smooth muscle cells (SMCs) expressing herpes simplex virus thymidine kinase (HSVtk) without systemic ganciclovir treatment (D) Arterial segment seeded with SMCs expressing HSVtk with systemic ganciclovir treatment. D Fig. 2. Histological sections from representative arterial segments (200 magnification). All specimens were obtained 21 days after balloon injury. (A) Uninju-red carotid artery (B) Balloon-injured carotid artery (C) Arterial segment seeded with smooth muscle cells (SMCs)expressing herpes simplex virus thymidine kinase (HSVtk) without systemic ganciclovir treatment (D) Arterial segment seeded with SMCs expressing HSVtk with systemic ganciclovir treatment. 983

장 일정한 손상이 가고 평활근 세포의 이식도 중앙 부 외로 더욱 심한 협착을 보였다. 풍선 손상을 가한 후 위에 이루어지기 때문에, 경동맥 중앙부위의 가장 협착 HSVtk 유전자를 가지는 혈관 평활근 세포를 이식하고, 이 심한 부위를 손상을 가하지 않은 좌측 경동맥을 기 이식 1주째부터 2주간 전신적 ganciclovir 치료를 시 준으로 분석하였다.

풍선 손상을 가한 후 HSVtk 유전자를 가지는 혈관 인 혈관 협착이 사라졌고, 좌측 경동맥을 기준 혈관으 평활근 세포를 이식하였으나 ganciclovir 치료를 시행 로 하였을 때 85±3% 내경을 보였다(Figs. 4 and 5).

01, Fig. 5). X-Gal 염색 본 실험에서 사용되는 유전자 이식 방법이 유전자 이 식을 성공적으로 할 수 있는 방법이라는 것을 검정하기 위하여, HSVtk 유전자 대신에 β-galactosidase 유전 Fig. 3.

magnification). There were inflammatory cell infiltrations at the dependent portion of the artery on the left side of the figure.

8 장 일정한 손상이 가고 평활근 세포의 이식도 중앙 부 외로 더욱 심한 협착을 보였다. 풍선 손상을 가한 후 위에 이루어지기 때문에, 경동맥 중앙부위의 가장 협착 HSVtk 유전자를 가지는 혈관 평활근 세포를 이식하고, 이 심한 부위를 손상을 가하지 않은 좌측 경동맥을 기 이식 1주째부터 2주간 전신적 ganciclovir 치료를 시 준으로 분석하였다. 풍선 손상만 가하고 유전자 치료를 행한 유전자 치료군(n 5)의 우측 경동맥은 풍선 손상 시행하지 않은 대조군(n 5)의 우측 경동맥은 좌측 경 만 준 대조군이나 풍선 손상 후 HSVtk 유전자를 가지 동맥을 기준 혈관으로 하였을 때 내경은 65±5%이었 는 혈관 평활근 세포 이식만 한 군에서 보이던 국소적 다. 풍선 손상을 가한 후 HSVtk 유전자를 가지는 혈관 인 혈관 협착이 사라졌고, 좌측 경동맥을 기준 혈관으 평활근 세포를 이식하였으나 ganciclovir 치료를 시행 로 하였을 때 85±3% 내경을 보였다(Figs. 4 and 5). 하지 않은 군(n 3)의 우측 경동맥은 내경은 40% 내 풍선 손상을 가한 후 HSVtk 유전자를 가지는 혈관 평활근 세포를 이식하고, 이식 1주째부터 2주간 전신 적 ganciclovir 치료를 시행한 유전자 치료군(n 5)의 우측 경동맥 직경(85±3%)은 풍선 손상만 준 대조군 의 경동맥 직경(65±5%)보다 유의하게 커져 있었다 (p< 0.01, Fig. 5). X-Gal 염색 본 실험에서 사용되는 유전자 이식 방법이 유전자 이 식을 성공적으로 할 수 있는 방법이라는 것을 검정하기 위하여, HSVtk 유전자 대신에 β-galactosidase 유전 Fig. 3. The magnified histological section from arterial segment seeded with smooth muscle cells (SMCs) expressing herpes simplex virus thymidine kinase (HSVtk) with systemic ganciclovir treatment (400 magnification). There were inflammatory cell infiltrations at the dependent portion of the artery on the left side of the figure. A B 자를 표적 유전자로 사용하고 쥐의 혈관평활근세포를 대상세포로 하는 retroviral vector system을 구축하 였다. β-galactosidase 유전자를 가진 혈관 평활근 세포를 손상된 쥐 경동맥 이식하여 그 유효성을 수술 C Fig. 4. Representative carotid arteriograms. Right carotid arteries were balloon-injured. All were obtained 21 days after balloon injury. (A) Balloon injury only (B) Smooth muscle cells (SMCs) expressing herpes simplex virus thymidine kinase (HSVtk) implantation only (C) SMCs expressing HSVtk implantation with systemic ganciclovir treatment. 984 Korean Circulation J 1998;28(6):

Histochemical staining for -galactosidase activity of the injured right rat carotid artery seeded with smooth muscle cells SMCs expressing -galactosidase. It was obtained 7 days after balloon injury.

9 Fig. 5. Balloon-injured carotid artery luminal diameter comparing with the uninjured artery. A Balloon injury only B Smooth muscle cells expressing herpes simplex virus thymidine kinase LtkSN / SMC transplantation with systemic ganciclovir treatment. Fig. 6. Histochemical staining for -galactosidase activity of the injured right rat carotid artery seeded with smooth muscle cells SMCs expressing -galactosidase. It was obtained 7 days after balloon injury. Gross specimen includes the aortic arch and both common carotid arteries. Intense blue staining is found at the upper part of the injured right common carotid artery. One scale of the ruler at the lower part of the figure is 0.5 cm. Fig. 7. The histological section from the injured right carotid artery seeded with smooth muscle cellssm- Cs expressing human placental alkaline phosphatase 40 magnification. It was obtained 7 days after balloon injury. Alkaline phosphatase reaction product stains black. Note asymmetric distribution of the labeled cells and the presence of unlabelled cell in the intima. 고 찰 985

10 986 Korean Circulation J 1998;286:

11 987

12 요약 연구배경 : 988 방법 : 결과 : 결론 : 중심단어 REFERENCES 1) Fischman DL, Leon MB, Baim DS, Schatz RA, Savage MP, Penn I, et al. A randomized comparison of coronarystent placement and balloon angioplasty in the treatment of coronary artery disease. N Engl J Med ) Serruys PW, De Jegere P, Kiemeneij F, Macaya C, Rutsch W, Heyndrickx G, et al. A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. N Engl J Med ) King SB III. Angioplasty from bench to bedside to bench. Circulation Korean Circulation J 1998;286:

13 4) Safian RD, Gelbfish JS, Erny RE, Schnitt SJ, Schmidt DA, Baim DS. Clinical, angiographic, and histological findings and observations regarding potential mechanisms. Circulation ) Post MJ, Borst C, Kuntz RE. The relative importance of arterial remodeling compared with intimal hyperplasia in lumen renarrowing after balloon angioplasty. Circulation ) Mintz GS, Popma JJ, Pichard AD, Kent KM, Satler LF, Chiu WS, et al. Arterial remodeling after coronary angioplasty A serial intravascular ultrasound study. Circulation ) Ross R. The pathogenesis of atherosclerosis An update. N Engl J Med ) Painter JA, Mintz GS, Wong C, Popma JJ, Pichard AD, Kent KM, et al. Serial intravascular ultrasound studies fail to show evidence of chronic Palmatz-Schatz stent recoil. Am J Cardiol ) Gordon PC, Gibson M, Cohen DJ, Carrozza JP, Kuntz RE, Baim DS. Mechanisms of restenosis and redilatation within coronary stents. J Am Coll Cardiol ) Anderson WF. Human gene therapy. Science ) Miller AD. Human gene therapy comes of age. Nature 1992; ) Mulligan RC. The basic science of gene therapy. Science ) Chang MW, Barr E, Seltzer J, Jiang YQ, Nabel GJ, Nabel EG, et al. Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science ) Chang MW, Ohno T, Gordon D, Lu MM, Nabel GJ, Nabel EG, et al. Adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene inhibits vascular smooth muscle cell proliferation and neointima formation following balloon angioplasty of the rat carotid artery. Molecular Medicine ) Guzman RJ, Hirschowitz EA, Brody SL, Crystal RG, Epstein SE, Finkel T. In vivo suppression of injuryinduced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci USA ) Bennett MR, Anglin S, McEwan JR, Jagoe R, Newby AC, Evan GI. Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides. J Clin Invest ) Oliver S, Babley G, Crumpacker C. Inhibition of HSV-tk transformed murine cells by nucleoside analogs 2-NDG and 2 -nor-gmp. Virology ) Takamiya Y, Short MP, Moolten FL, Fleet C, Mineta T, Breakefield XO, et al. An experimental model of retrovirus gene therapy for malignant brain tumors. J Neurosurg ) Ram Z, Walbridge S, Shawker T, Culver KW, Blease RM. The effect of thymidine kinase transduction and ganciclovir therapy on tumor vasculature and growth of 9L gliomas in rats. J Neurosurg ) Freeman SM, Abboud CN, Whartenby KA, Packman CH, Koeplin DS, Moolten FL, et al. The bystander effect Tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res ) Bi WL, Parysek LM, Warnick R, Stambrook PJ. In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSVtk retroviral gene therapy. Hum Gene Ther ) Eglitis MA, Anderson WF. Retroviral vectors for introduction of genes into mammalian cells. Biotechnique ) Salmons B, Gunzburg WH. Targeting of retroviral vectors for gene therapy. Hum Gene Ther ) Clowes AW, Reidy MA, Clowes MM. Kinetics of cellualr proliferation after arterial injury.. Smooth muscle growth in the absence of endothelium. Lab Invest ) Clowes MM, Lynch CM, Miller AD, Miller DG, Osbome WRA, Clowes AW. Long-term biological response of injured rat carotid artery seeded with smooth muscle cells expressing retrovirally introduced human genes. J Clin Invest ) Sambrook J, Fritsch EF, Maniatis T ed. Molecular cloninga laboratory manual. Cold Springs Harbor Lab Press, NY ) Hock RA, Miller AD, Osborne WRA. Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines. Blood ) Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques ) Miller AD, Bultimore C. Redesign of retrovirus packaging cell lines to avoid recombination to helper virus production. Mol Cell Biol ) Lynch CM, Clowes MM, Osborne WRA, Clowes AW, Miller AD. Long-term expression of human adenosine deaminase in smooth muscle cells of rats A model for gene therapy. Proc Natl Acad Sci USA ) Nabel EG, Pompili VJ, Plautz GE, Nabel GJ. Gene transfer and vascular disease. Cardiovasc Res ) Nabel EG. Gene therapy for cardiovascular disease. Circulation ) Verma IM, Somia N. Gene therapy-promises, problems and prospects. Nature ) Kim ST, Kim DW, Oh TG, Ahn HY, Kim YG. Gene therapy using retroviral vector containing rat erythropoietin. Korean J Hematology ) Ohno T, Gordon D, San H, Pompili VJ, Imperiale MJ, Nabel GJ, et al. Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science ) Barbee RW, Stapleton DD, Madras DE, Re RN, Murgo JP, Davenport WD, et al. Retroviral suicide vector does not inhibit neointimal growth in a porcine coronary model of restenosis. Biochem Biophys Res Comm ) Chen SJ, Chen YF, Miller DM, Li H, Oparil S. Mithramycin inhibits myointimal proliferation after balloon injury of the rat carotid artery in vivo. Circulation

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract