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Korean Journal of Obstetrics & Gynecology 2001;44(6):1137-1144.
Published online June 1, 2001.
Adenovirus and Lipofectin Mediated p53 Gene Transfection Efficiency in Human Cervical Cancer Cell Lines and Xenografted Nude Mouse.
Kyeong A Yeo, Jae Eun Jung, Hye Young Lee, Bang Hyon Lee, Tae Hyung Kim, Byeung Soo Cheung, Min Suk Rho, Joon Mo Lee, Sung Eun Namkoong, Yong Seok Park, Chong Kook Kim, Woong Shick Ahn
1Department of Obstetrics and Gynecology, College of Medicine, The Catholic university of Korea.
2Research Institute of Cancer, Catholic Research Institute of Medical Science.
3Department of medical technology, College of Health Science, Yonsei university.
4College of Pharmacy, Seoul National University
Abstract
BACKGROUND: The classical treatment of the cervical cancer is surgery, radiotherapy, chemotherapy. Even though the improvement of treatment successful rate, conventional therapy has some limitations. Recent cutting edge of cancer therapy has been developed in gene level including understand the biological characteristics of the cancer cells, enhance the human immune response, suppress the cancer cell proliferation. Therefore, the gene therapy is proposed to new treatment strategy. PURPOSE: The transfection efficiency of cervical cancer cell lines and cervical cancer cell line xerografted nude mouse was investigated by transfection of liposome and infection of adenovirus mediated suppressor(p53) and reportor(LacZ) gene. METHOD: The cervical cancer cell lines was used in this study were CaSki, SiHa (HPV16 positive, wild type p53 gene), HeLa, HelaS3(HPV18 positive, wild type p53 gene) and C33A, HT3(HPV negative, mutant p53). Direct plasmide and AdCMVp53 gene transfection was performed by using liposome system (pRcCMVLacZ / lipofectin, FuGene 6, Ca-phosphate). LacZ gene was used as the reportor gene for the transfection efficiency evaluation. Expression of p53 in cell lines and tumor tissue was confirmed by western blot and immunohistochemical staining. Xenografted nude mouse of SiHa cell line was infected by AdCMVp53 and AdCMVLacZ. Transfection efficiency was observed by same as above. RESULTS: In cervical cancer cell lines, gene transfection using liposome system(pRcCMVLacZ/lipofectin, FuGene 6, Ca-phosphate)revealed different transfection efficiency, especially pRcCMVLacZ in Fugene 6 showed 18-40% of high transfection efficiency in 6 cervical cancer cell lines by X-gal staining and AdCMVp53 showed 95-98% of the high transfection efficiency in HeLa, C33A. AdCMVp53 was significantly expressed at 2-5days after injection xenografted nude mouse on the western blot and transfection efficiency was 19.79+/-5.36, 26.26+/-11.69, 14.77+/-3.98,15.99+/-6.43%(day1-5). AdCMVLacZ were found to immuno- histochemistry analysis, in vivo transfection efficiency was 61.26+/-4.66,59.63+/-9.12, 29.46+/-14.33, 31.73+/- 22.64%(day 1-5) atx200 and 88.65+/-8.65, 70.85+/-20.94, 40.75+/-25.44, 48.21+/-10.97% (day 1-5) atx400. CONCLUSION: As a results, adenovirus-mediated transfection efficiency was higher in vivo experiment compared to cell lines. These high efficiency of adenovirus-mediated suppressor gene(p53) could become a significant meaningful data gene therapy strategy both transgenic mice and cervical cancer cell lines.
Key Words: p53, AdCMVp53, cervical cancer, gene therapy. immunohistochemistry
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