인유두종 바이러스의 발암성 단백질의 발현을저해하는 Trans Attack 항암성 유전자 백신의 개발
Development of Trans Attack Anticancer Gene Vaccine Interactin HPV Oncoprotein Expression Inhibition
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목적 : 자궁경부암을 일으키는 것으로 알려진 인유두종 바이러스의 종양단백질 E6을 직접 저해하기 위하여 아데노 연관바이러스 (adeno-associated virus; AAV)의 Rep78유전자를 이용, E6의 작용을 차단함으로써 자궁경부암 세포성장의 억제에 이용하고자 하였다. 연구 방법 : 아데노 연관바이러스에서 유래된 Rep78유전자를 클로닝하여 pcDNA3.1/V5/His-Topo/Rep78벡터를 제작, 이를 자궁경부암 세포주에 이입시켰다. Rep78 유전자 이입 후 인유두종 바이러스에 의한 종양단백질 E6 mRNA의 발현을 보았으며 자궁경부암 세포성장 억제를 측정하였다. 결과 : CaSki, SiHa 세포주에서 Rep78 벡터에 의한 이입율은 30-60% 이었으며, 이입 24시간 후 제일 높은 발현이 나타났다. Rep78의 발현 후 세포 성장억제 실험 결과 자궁경부암 CaSki와 HeLa에서는 4일째에 세포성장을 억제하는 것을 보였으나, 그 외의 자궁경부암 세포주에서는 뚜렷한 차이를 보이지 않았다. 결론 : Rep78에 의한 세포내의 높은 이입율과 발현에도 불구하고 자궁경부암 세포주의 성장저해 효과는 나타나지 않았다. 아데노 연관바이러스를 이용한 자궁경부암의 치료에 있어서 Rep78의 발현을 오랜 시간 동안 지속시키는 전략이 필요하다.
Abstract
Objective : Adeno-associated virus Rep 78 protein is known to inhibit the promoter site of several onco-genes and viral gene, including the human papillomavirus type 16 E6 transforming genes. In this study, we investigated AAV Rep 78 mediated inhibition of HPV 16 E6 promotor activity. Methods : pcDNA3.1/V5/His-Topo vector, cloned by AAV mediated Rep 78, is transfected into cervical cancer cell line (Caski). After that, we confirmed HPV16 derived E6 expression and cell growth inhibition. Results : Transfection rate of Rep78 GFP-vector, approximately from 30 to 60 per-cent, is highly expressed at first day. But E6 expression is lower at this day. The growth of CaSki and HeLa cervical cancer cell lines was inhibited by Rep78 (p<0.05). But, the other cervical cancer cells were unaffected by Rep78 transfection. Conclusion : In spite of the high Rep78 transfection efficiency and expression rate, we could not show the cervical cancer cell growth inhibition. In our data, long term expression of Rep78 strategy is needed for cervical carcinoma gene therapy using adeno-associated virus vector.
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