서론 978 Korean Circulation J 1998;286:

Original Articles Korean Circulation J 1998;286:977-989 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;286:977-989 KEY WORDSGene therapy Restenosis HSVtk gene. 977

서론 978 Korean Circulation J 1998;286:977-989

방법 LtkSN의제작 979

재조합 retrovirus의제작 980 쥐혈관평활근세포배양및 Retroviral vector 감염 HSVtk 유전자치료모델 Korean Circulation J 1998;286:977-989

유전자이식검정 981

실험동물의분류 통계적분석 982 결과 조직학적소견 경동맥조영술 Korean Circulation J 1998;286:977-989

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 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):977-989

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

986 Korean Circulation J 1998;286:977-989

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