단순 헤르페스 제1형 티미딘 키나제 유전자 이입 간암세포주에서 방사표지 IVDU와 IVFRU의 섭취 평가 = In Vitro Uptakes of Radiolabeled IVDU and IVFRU in Herpes Simplex Virus Type-1 Thymidine Kinase (HSVl-tk) Gene Transduced Morris Hepatoma Cell Line

국문 초록 (Abstract)

목적: 단순 헤르페스 제 1형 티미딘 키나제(herpes simplwx virus type 1 thymidines kinase gene: HSV1-tk)는 GCV와 함께 유전자치료의 한 방법으로서 가장 활발하게 연구되어왔으며, HSV1-tk 효소에 대한 다양한 기질들이 연구되어서 이를 보고 기질로 한 비침습적인 HSV1-tk 유전자 영상시스템에서 가장 널리 사용되고 있다. 본 연구에서는 보고기질로서 방사성 요오드가 표지된 5-iodovinyl-2-deoxyuridine (IVDU) and 5-Iodovinyl-2-fluoro-2-deoxyuridine(IVFRU)를 보고 기질로 하여 HSV1-tk 유전자 영상시스템에서의 유용성을 확인하고자 하였다. 대상 및 방법: HSV1-tk 유전자 영상을 위하여 HSV1-tk 유전자를 레트로 바이러스 벡터를 이용하여 Morris hepatoma 세포주(MCA)-tk에 이입한 세포주를 제조하였으며, HSV1-tk 유전자의 발현을 확인하기 위하여 Northern blotting과 Western Blotting을 시행하였다. 대조세포주인 MCA와 제조된 MCA-tk 세포주에 방사표지 IVDU와 IVFRU를 이용하여 480분까지 세포내 섭취율을 평가하였으며, 또한 MCA-tk 세포주의 백분율을 증가시키면서 이에 따른 IVDU 와 IVFRU의 섭취율을 평가함으로서 섭취율과 세포수와의 상관관계를 평가하였다. 결과: MCA-tk 세포주에서 HSV1-tk의 mRNA의 발현과 HSV1-TK 단백질의 발현을 확인하였다. 방사성 요오드 표지 IVDU와 IVFRU 모두는 MCA 세포주에서는 아주 낮은 섭취율을 나타내었으며, MCA-tk 세포주에서는 모두 증가된 섭취를 보였다. IVDU가 480분에서 IVFRU보다 4배 높은 섭취를 나타내었다(p<0.01). 방사표지 IVDU와 IVFRU 모두에서 MCA-tk의 백분율의 증가에 따라서 직선적인 상관관계(R^(2)>0.96)를 나타내었다. 결론: 방사선 요오드 표지 IVDU와 IVFRU는 HSV1-tk 유전자가 이입된 간암세포주에서 모두 특이적인 높은 섭취율을 나타내고 직선적인 상관관계가 나타나서 두 기질 모두 HSV1-tk 유전자 영상시스템에서 보고 기질로서 유용하게 사용될 수 있을 것으로 기대된다.

다국어 초록 (Multilingual Abstract)

Purpose: The herpes simplex virus type 1 thymidine kinase gene(HSV1-tk) is 문 attractive candidate as a reporter gene in noninvasive reporter gene monitoring system. The HSV1-tk gene was chosen as a reporter gene, because it has been extensively studied, and there are appropriate reporter probes, substrates of HSV1-tk gene product, to apply for HSV1-tk gene imaging. We used radiolabeled 5-iodovinyl-2-deoxyuridine (IVDU) and 5-iodovinyl-2-fluoro-2-deoxyuridine(IVFRU) as reporter probes for HSV1-tk gene monitoring system. Materials and Methods: We prepared HSV1-tk gene transduced Morris hepatoma cell line usting retroviral vector, MOLTEN containing HSV1-tk gene. And we confirmed the HSV1-tk gehe expression by Northern blotting and Western blotting. We compared in citro uptakes of radioiodinated ICVU and IVFRU to monitor HSV1-tk hene expression in Morris hepatoma cell line (MCA) and HSV1-tk gene tranduced MCA (MCA-tk) cells until 480 minutes. We also performed correlation analysis between percantage of HSV1-tk gene tranduced MCA cell ％ (MCA-tk％) and uptakes of radiolabeled IVDU or IVFRU. Results: MCA-tk cell expressed HSV1-tk mRNA and HSV1-TK protein. Two compounds showed minimal uptake in MCA, but increased uptake was observed in MCA-tk. IVDU showed 4-fold higher accumulation than IVFRU at 480 min in MCA-tk (p<0.01). Both IVDU and IVFRU uptake were linearly correlated (R^(2)>0.96) with increasing MCA-tk％. Conclusion: The radiolabeld IVDU and IVFRU showed higher specific accumulation in retrovirally HSV1-tk gene transfected Morris hepatoma cell line. Both IVDU and IVFRU could be used as good substrates for evaluation of HSV1-tk gene expression.

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