A Basic Study of the Replication of Human Papillomavirus Type16 Episomal DNA from a Cervical Carcinoma Cell Line CC7T/VGH

• Main Authors: Deng-Yi Lin, 林登藝
• Other Authors: Ming-Liang Li
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/50895382668459957644

碩士 === 國立師範大學 === 生物學研究所 === 83 === HPV16 基因體常在子宮頸癌檢體中被檢出，具有游離(episome)及插入兩種 形式，但其無法在體外培養，癌細胞衍出的細胞株又多只具插入形式HPV16 DNA使其複製和寄主同步，故其複製研究不易。1980年榮總胡承波博士建立 一子宮頸癌細胞株-CC7T/VGH，同時具有游離及插入形式HPV16 DNA ，其游 離基因體之E1、E7及L1皆發生突變，為研究HPV16複製的理想材料，本實驗 即將直線狀之重組HPV16 prototype或/及由CC7T/VGH選殖之游離基因體DNA 轉染至另一子宮頸癌(HeLa)或咽喉癌(HEp-2)細胞株中觀察其複製情形，以 判別CC7T中HPV16 episome之存在是否為episome DNA 本身的突變所造成。 實驗結果發現轉染五天內，細胞皆無明顯生長遲滯情形，形態亦正常，而 不論轉染重組HPV16 prototype、游離基因體DNA或二者，HPV16 DNA在HeLa 及HEp-2細胞中的量皆隨時間的增加而逐漸減少，並無複製現象，故HPV16 游離基因體本身的變異應不是其穩定存在CC7T/VGH細胞中的主因。將 CC7T/VGH抽取出來的低分子量DNA以限制酵素BamHI切割，可觀察到7.7kb的 HPV16游離基因體DNA片段及一段約3.1kb大小的DNA片段，此3.1kb片段經不 同限制酵素切割，得知其上至少有EcoRI、KpnI及PstI的切割區。 Human papillomavirus (HPV) type16 genome is usually detected from the biopsies of patients who carry cervical intraepithelial neoplasma (CIN). Both episomal and integrated forms of HPV16 genome have been found in these cells but viruses can not be produced when cultured in vitro. Most cell lines derived from CIN are found to have only integrated form of HPV16 DNA and that makes the integrated HPV16 DNA replicating synchronously with the host cell, thereby, it becomes difficult to study the independent replication of HPV16 DNA in this regard. In 1980, Dr. C. P. Hu established a cervical carcinoma cell line CC7T/VGH, which contains both episomal and integrated HPV16 DNA. The episomal DNA in the CC7T/VGH cell contains mutations in its E1, E7 and L1 open reading frames and can be a good system for comparative study the replication of HPV16 genome. We transfected the linear recombinant HPV16 prototype or episomal (cloned from CC7T/VGH)DNA into other cervical carcinoma (HeLa) or larynx carcinoma (HEp-2) cell lines to observe the consequence of transfected HPV16 genome . Our results showed that the morphology and growth rate did not change distinctively in all transfected cells. The amount of HPV16 DNA was gradually decreased after transfection for 5 days when recombinant HPV16 prototype or episomal DNA or both were tranfected. No any HPV16 DNA replication in HeLa or HEp-2 was shown which indicated that the mutation of episome might not play an important role for existence of episomal HPV16 DNA in the CC7T/VGH cell. We also observed an unusual DNA fragment from the BamHI restriction endonuclease digestion of low molecular weight DNA of the CC7T/VGH cell. The fragment is about 3.1kb and has EcoRI, KpnI and PstI recognition sites. The significant existence of this small fragment is currently under investigation.