CAJ | 학술논문

目的探讨转染与筛选技术获取稳定携带有HPV11基因组DNA的细胞的可能性.方法对含有pBR322.HPV11质粒的大肠杆菌培养扩增,然后进行质粒的提取与纯化,并用限制性内切酶BamH Ⅰ切割下HPV11全长基因.低熔点琼脂糖回收后,用T4 DNA连接酶进行线状DNA的自身环化,再与pIK-neo质粒、用Lipofectamine试剂共转染HaCaT细胞.通过G418筛选阳性细胞克隆,将阳性克隆细胞合并与培养扩增后,用FQ-PCR技术检测细胞内HPV11DNA的存在,用巢式RT-PCR技术检测HPV11E1^E4 mRNA的表达.结果 HPV11型基因组DNA转染至HaCaT细胞后,经G418筛选约2周,培养皿内即可见对G418抵抗的阳性细胞克隆出现,其形态与普通HaCaT细胞相似.用FQ-PCR在筛选后的HaCaT细胞中检测到HPV11DNA的存在,平均病毒DNA载量为(15.9±16.8)拷贝/细胞.传代3次后细胞内病毒DNA无丢失,载量为(23.9±1.1)拷贝/细胞.与未传代细胞相比,二者间差异无统计学意义(t=-0.822,P>0.05).巢式RT-PCR扩增产物经琼脂糖凝胶电泳后检测到HPV11 E1^E4 mRNA表达的特异性628 bp条带.结论 HPV11基因组DNA可通过脂质体转染法成功导入HaCaT细胞内,通过筛选可获得阳性细胞,且经3次传代后HPV11DNA仍然存在.
목적 탐토전염여사선기술획취은정휴대유HPV11기인조DNA적세포적가능성.방법 대함유pBR322.HPV11질립적대장간균배양확증,연후진행질립적제취여순화,병용한제성내절매BamH Ⅰ절할하HPV11전장기인.저용점경지당회수후,용T4 DNA련접매진행선상DNA적자신배화,재여pIK-neo질립、용Lipofectamine시제공전염HaCaT세포.통과G418사선양성세포극륭,장양성극륭세포합병여배양확증후,용FQ-PCR기술검측세포내HPV11DNA적존재,용소식RT-PCR기술검측HPV11E1^E4 mRNA적표체.결과 HPV11형기인조DNA전염지HaCaT세포후,경G418사선약2주,배양명내즉가견대G418저항적양성세포극륭출현,기형태여보통HaCaT세포상사.용FQ-PCR재사선후적HaCaT세포중검측도HPV11DNA적존재,평균병독DNA재량위(15.9±16.8)고패/세포.전대3차후세포내병독DNA무주실,재량위(23.9±1.1)고패/세포.여미전대세포상비,이자간차이무통계학의의(t=-0.822,P>0.05).소식RT-PCR확증산물경경지당응효전영후검측도HPV11 E1^E4 mRNA표체적특이성628 bp조대.결론 HPV11기인조DNA가통과지질체전염법성공도입HaCaT세포내,통과사선가획득양성세포,차경3차전대후HPV11DNA잉연존재.
Objective To explore if keratinocytes that stably maintain HPV11 genome can be obtained by transfection and selection methods. Methods Escherichia coil containing pBR322.HPV11 plasmid was cultured and amplified. Then the plasmid was extracted, purified and digested with BamH Ⅰ enzyme to release viral genome from the bacterial vector. After recovering from the low-melting point agarose gel by electrophoresis, the genome was self-circulated with T4 DNA ligase. The religated DNA was cotransfected with pTK-neo DNA into HaCaT keratinocytes using Lipofectamine reagent. After selection with G418 for 2 to 3 weeks, clonal and pooled cultures were expanded and analyzed. Fluorescent quantitative PCR (FQ-PCR) and nested reverse transcriptase PCR (nRT-PCR) were applied to detect HPV11 DNA and spliced HPV11 E1^E4 mRNA expression in the transfected cells. Results After the cotransfection of HPV11 genome into HaCaT keratinocytes and two-week selection,G418-resistant cell colonies were obtained with morphological features indistinguishable from normal HaCaT keratinocytes. As shown by FQ-PCR, HPV11 DNA was present in G418-selected HaCaT keratinocytes. The average viral DNA load capacity was 15.9±16.8 copies/cell in the primary culture of G418-selected HaCaT cells and 23.9±1.1 copies/cell in the third passage of the cells; there was no statistical difference between the two passages of cells (t=-0.822, P>0.05). nRT-PCR targeting HPV11 E1^E4 mRNA transcript produced a specific 628-bp fragment, which was shown by agarose gel electrophoresis. Conclusions Our data indicate that HPV11 genome can be successfully introduced into HaCaT keratinocytes by transfection and HPV11 DNA-positive cells can be obtained by G418 selection. Moreover, HPV11 DNA is still present in the third passage of transfected cells.