研究生: |
林登藝 Deng-Yi Lin |
---|---|
論文名稱: |
人類乳突瘤病毒第十六型(HPV16)游離基因體(Episome)在子宮頸癌細胞株 A Basic Study of the Replication of Human Papillomavirus Type16 |
指導教授: |
李銘亮
Li, Ming-Liang |
學位類別: |
碩士 Master |
系所名稱: |
生命科學系 Department of Life Science |
畢業學年度: | 83 |
語文別: | 中文 |
論文頁數: | 43 |
中文關鍵詞: | 人類乳突瘤病毒;游離基因體;複製 |
英文關鍵詞: | Human papillomavirus;Episome;replication;HPV16 |
論文種類: | 學術論文 |
相關次數: | 點閱:183 下載:0 |
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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.
Human papillomavirus (HPV) type16 genome is usually detected from