转导EB病毒转录的EBERs促进MDCK细胞的恶性化

Malignant transformation of madine-darby canine kidney (MDCK) epithelial cells by EBV encoded small RNAs (EBERs)

关键词:  EBERs  MDCK  分散活性  基质非依赖性  癌化

摘要:

通过转导EB病毒 (Epstein-Barr Virus, EBV) 转录的核内小RNA (EBV encoded small RNAs, EBERs) 探讨其对上皮细胞株MDCK细胞生长特性的影响。［方法］ 用聚合酶链反应 (polymerase chain reaction, PCR) 技术从EB病毒阳性的鼻咽癌 (nasopharyngeal carcinoma, NPC) 由来的NPC-KT细胞中扩增出EBERs片段。为了在细胞内大量表达EBERs, 利用分子生物学技术，制作了EBERs片段的5次重复排列的5EBERs和10次重复排列的10EBERs。分别插入pCEP4载体质粒,并导入MDCK细胞内。 利用Northern Blot法确定细胞内EBERs的表达后, 对其在培养中的细胞生长特性和软琼脂中的细胞克隆形成进行了观察。［结果］ 在培养中大量表达EBERs的MDCK细胞之间丧失了细胞间的相互连接,细胞呈纺锤状，并在软琼脂中形成大量细胞克隆。而非表达EBERs的MDCK细胞的对照组中没有发现细胞生长特性的改变和大量细胞克隆形成。［结论］ EBERs的大量表达可以造成MDCK细胞的分散活性和基质非依赖性。提示EBERs在上皮细胞株MDCK细胞中有癌化作用，从而推论EBERs在同样源于上皮细胞的鼻咽癌中也可能有癌化作用。另外, 表达EBERs的MDCK细胞的建立为进一步研究EBERs的作用提供了一个有用的上皮细胞模型。

Abstract:

To observe the growth characteristics of the EBERs-transfected MDCK cells, through generated overexpression of EBERs in MDCK cells. ［Methods］ EBERs fragment were amplified by PCR from EBV-positive NPC-KT cell DNA. Using molecular biologic technique, we constructed the EBERs-expressing plasmids,which contained , 5 or 10 tandem repeats of the EBERs. The MDCK cells were transfected with those plasmids, respectively. We demenstrated the expression of EBERs in EBERs-transfected MDCK cells by Northern blot analysis, and compared the growth characteristics in culture and colony formation in soft agar of Hygr- and EBERs-transfected MDCK cells. ［Results］ 10EBERs-transfected MDCK cells lost cell-to-cell contacts and acquired long spindle-shape morphology in culture and formed colonies in soft agar. However, parental and Hygr-transfected MDCK cells scarcely did.The authors established 10EBERs-transfected MDCK cells, which overexpressed the EBERs and show that it has a cattering activity in culture and a role in anchorage-independent growth in soft agar. ［Conclusions］ These results indicat that EBERs has a role of malignant transformation in MDCK cells. However, the 10EBERs-transfected MDCK cell provides a useful epithelial model for stud ying the function of EBERs.