Immortalization of primary human smooth muscle cells.
Immortalization of primary human smooth muscle cells.
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原代人平滑肌细胞的永生化。
DOI：
10.1073/pnas.89.4.1224
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发表时间：
1992
影响因子：
11.1
通讯作者：
McDougall,JK
中科院分区：
文献类型：
--
作者：
Perez-Reyes,N;Halbert,CL;Smith,PP;Benditt,EP;McDougall,JK
使用含有人乳头瘤病毒16型的E6和E7开放阅读框（ORF）的嗜中性重组逆转录病毒构建体使原代人主动脉和子宫肌层平滑肌细胞（SMC）永生化。与非永生化对照细胞相比，表达E6/E7 ORF的SMC具有相当高的生长速率，并且在长期传代中没有显示出衰老的迹象。本研究中获得的第一个SMC系已在连续组织培养中保持超过1年（超过180个群体倍增）。通过间接免疫荧光测定，永生化SMC具有减小的细胞大小和减少的肌肉特异性α-肌动蛋白丝含量。Southern印迹分析表明E6/E7 ORF在逆转录病毒感染的细胞中稳定整合，放射免疫沉淀证实了E6和E7基因的持续表达。SMC系的细胞遗传学研究显示，除了子宫肌层克隆系之外，其基本上为二倍体群体，子宫肌层克隆系在后期传代（大于125倍体）时变为非整倍体。这些细胞系在裸鼠中不具有致瘤性。
Primary human aortic and myometrial smooth muscle cells (SMCs) were immortalized using an amphotropic recombinant retroviral construct containing the E6 and E7 open reading frames (ORFs) of human papillomavirus type 16. The SMCs expressing the E6/E7 ORFs have considerably elevated growth rates when compared with nonimmortalized control cells and show no signs of senescence with long-term passage. The first SMC line derived in this study has been maintained in continuous tissue culture for greater than 1 year (greater than 180 population doublings). The immortalized SMCs have decreased cell size and decreased content of muscle-specific alpha-actin filaments as determined by indirect immunofluorescence. Southern blot analysis has demonstrated the stable integration of the E6/E7 ORFs in the retrovirally infected cells, and radioimmunoprecipitation has confirmed the continued expression of the E6 and E7 genes. Cytogenetic studies of the SMC lines have revealed essentially diploid populations except for the myometrial clonal line, which became aneuploid at late passage (greater than 125 doublings). These cell lines were not tumorigenic in nude mice.