KISEP Experimental Researches J Korean Neurosurg SocSupplement Ⅰ 3S13-S19, 21 아데노바이러스 Cytosine Deaminase/Thymidine Kinase 융합유전자의항종양효과 * 김영우 최재영 장진우 박용구 정상섭 Abstract Antitumor Effect of an Adenoviral Cytosine Deaminase/Thymidine Kinase Fusion Gene in C6 Glioma Cells Young Woo Kim, M.D., Jae Young Choi, M.D., Jin Woo Chang, M.D., Ph.D., Yong Gou Park, M.D., Ph.D., Sang Sup Chung, M.D., Ph.D. Department of Neurosurgery, Yonsei University College of Medicine, Seoul, Korea bjective Methods Result Conclusion- KEY WORDS 서론 J Korean Neurosurg SocSupplement Volume 3December, 21 S13
- - - 대상및방법 1. 세포주및재조합아데노바이러스 - - - - 1) ad-cd/tk 2) ad-δe1 - - 3) pδe1spla, PWZLneoCDglyTK S14 J Korean Neurosurg SocSupplement Volume 3December, 21
4) Bgl II, EcoRV 2. X-5-Bromo-4-chloro-3-indolyl-13-D-galactoside(X- Gal) staining - 3. Immunoblotting analysis - - - - - 4. CD/TK 융합유전자와관련된세포독성의실험연구 - - 5. 통계분석 결 1. C6 glioma 세포에서재조합아데노바이러스의감염효율과 CD/TK 융합유전자의발현 과 J Korean Neurosurg SocSupplement Volume 3December, 21 S15
아데노 바이러스 Cytosine Deaminase/Thymidine Kinase 융합 유전자의 항 종양효과 Uninfected MOI=1 MOI=2 백질을 확인할 수 있었고 이 사실은 ad-cd/tk 감염된 후 Fig. 1. The transduction efficiency of recombinant adenovirus ion C6 glioma cells. Transduction efficiency was determined by examining expression of β-galactosidase (LacZ) after infecting the cells with ad-β-galactosidase. Dark staining represented successful β-galactosidase gene expression in the cells. The expression of β-galactosidase in cultured C6 glioma cells. The cells were infected with ad-β-galactosidase at MOI of 1, 2 or 5 and then stained with X-gal. Two days post-infection. MOI=5 MOI 1 1 1.1 1 CD/TK 융합 유전자의 표현이 적절히 되었음을 의미한다 (Fig. 2). Fig. 2에서 보는 바와 같이 같은 수준의 α-actin 이 각각의 세포군에서 측정되었는데 이것은 각각의 세포군에 같은 양의 단백질이 생성되었음을 의미한다. 293 세포는 CD/TK를 발견하는데 상당히 낮은 중복감염지수에서도 가 (KD) 97.4 66-46 - actin 능하였고 CD/TK가 표현되는 양은 특이한 차이가 없었다. C6 glioma 세포에서는 같은 수준의 CD/TK를 표현하기 위해서는 보다 높은 중복 감염지수로 감염시켜야 하였다 (.1-1 MOI 293세포 대 1 MOI C6 뇌교종 세포) CD/ TK 표현에 있어서 MOI를 증가시키는 것에 대한 긍정적인 효과는 C6 glioma 세포에서 관찰되었다. 높은 중복 감염지 CDTK C6 cells 293 cells Fig. 2. The expression of CD/TK in C6 glioma or 293 cells. The cells were infected with ad-cd/tk at designated MOIs and the equal amount of proteins was subjected to 6% SDS-PAGE. The CD/TK fusion proteins were visualized by immunoblotting analysis with monoclonal antibody to TK. The position of fusion protein CD/TK was indicated by arrow. 수로 감염시킨 세포에서는 면역 반응을 보인 CD/TK 단백 질이 많이 관찰되었다(Fig. 2). 또 1 MOI로 감염시킨 것과 1 MOI로 감염시킨 것을 비교했을 때 CD/TK 표 현되는 양은 큰 차이가 없었고 5 MOI가 넘을 경우 바이 러스 감염의 포화효과를 나타내었다. MOI 2에서 5-FC(1ug/ml) GCV(.1mg/ml)의 낮은 약물농도에서 비슷한 세포 독성을 나타내었다. 그러나 세 포독성의 범위는 5ug/ml 5-FC,.5ug/ml GCV에서 포 화 상태에 이르렀다. 또 Fig. 3C에서 보는 바와같이 GCV 2. ad-cd/tk 감염후 전구약물의 세포독성에 대한 실험 연구 1 MOI에서는 바이러스 자체는 어느정도는 세포독성을 와 5-FC로 동시에 치료하는 병합요법은 보다 큰 세포독 나타냈지만 전구약물 혼합요법으로는 세포 독성 효과를 관 ug/ml GCV는 현저한 세포 독성을 나타내지 못하였다(1 찰할 수 없었다. 그러므로 CD/TK의 표현이 전구약물(5- ug/ml 5-FC의 경우 종양세포가 줄지않았고.1ug/ml GCV FC, GCV)과 병합치료를 시행하였을 경우 세포독성을 일 경우 28±1%가 종양세포가 줄었다.). 그러나 같은 농도 으키는 지의 여부를 알기 위해서는 C6 glioma세포를 MOI 의 1ug/ml 5-FC와.1ug/ml GCV를 같이 병합해서 사 1~2에서 ad-cd/tk 또는 ad-δe1으로 감염시킨 용했을 때 53±8%의 종양세포가 감소되는 효과가 있었다. 후에 5-FC와 GCV 약물을 각각 다른 농도에서 그리고 혼 각각 다른 정도의 전구약물 농도와 MOI에서도 5-FC와 합농도에서 관찰하였다. 3ug/ml 이상의 농도에서 5-FC GCV를 병합해서 사용했을 때 동일하게 향상된 세포독성 는 현저한 세포독성을 나타내었고 MOI 1에서.3ug/ml 효과를 보인 반면에 단일 전구 약물로 치료했을 경우 세포 이상의 농도에서 GCV는 현저한 세포독성을 나타내었다. 독성에 있어서 포화 효과를 보였다(Fig. 3C). S16 성을 나타내었다. 1 MOI일 때 1ug/ml 5-FC 또는.1 J Korean Neurosurg Soc(Supplement I)/ Volume 3/ December, 21
Viability% A 16 14 12 1 8 6 4 2.2.4.6.8 1 GCVg/ml Viability% B 16 14 12 1 8 6 4 2 2 4 6 8 1 5-FCg/ml 14 14 12 12 Viability% 1 8 6 4 Viability% 1 8 6 4 2 2 C Ad-E1 MOI=1 Ad-E1CD/TK Ad-E1 MOI=2 Ad-E1CD/TK Fig. 3. In vitro cytotoxicity of ad-cd/tk combined with GCV or 5-FC treatment in C6 glioma cells. The cells infected with ad-cd/tk were exposed to GCV or 5-FC alone or in combined treatments after plating into 96 wells. The cell survival rate was determined by crystal violet staining Six days after infection. The mean values of at least three independent experiments are represented and standard errors are represented by error bars. ASensitivity to GCV of the cells transduced with ad-cd/tk. The cells were infected with ad-cd/tk at MOI of 1 or 2. As a control group, the cells were mock-infected or infected with ad- E1 at MOI of 2. BSensitivity to 5-FC of the cells transduced with ad-cd/tk. The data were represented as Fig. 3A. CAdditive effect of GCV and 5-FC treatment. The cells were treated with 5-FC of 1g/ml, GCV of.1g/ml or both after transduction with ad-e1 or ad-cd/tk at MOI of 1leftor 2Right 고찰 - J Korean Neurosurg SocSupplement Volume 3December, 21 S17
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