NIH3TCD40L稳定细胞系构建及在EBV转化B淋巴细胞中的应用.PDF

网络出版时间：2018-05-21 16:46:07 网络出版地址：/kcms/detail/31.2035.Q1645.014.html 中国细胞生物学学报 Chinese Journal of Cell Biology 2018, 40(5): 768–774 DOI: 10.11844/cj cb.2018.05.0316 NIH3T3-CD40L稳定细胞系构建及在EBV 转化B淋巴细胞中的应用 刘 玺 赵 烨 许崇凤 段子渊* ( 中国科学院遗传与发育生物学研究所, 遗传资源研究中心, 北京 100101) 摘要 通过脂质体法转染pCMV6-CD40L重组质粒和G4 18阳性克隆筛选, 构建了稳定表达 CD40L的NIH3T3细胞系, 在RNA和蛋白质水平进行了功能鉴定, 对其作为饲养细胞在体外B淋巴 母细胞系(B lymphoblastoid cell lines, B-LCLs)培养和在EBV转化B细胞中的作用进行了实验验证。 结果表明, NIH3T3-CD40L细胞系不仅可以提高体外低密度B-LCLs存活率, 而且可以降低成功建立 B-LCLs所需的细胞密度, 促进其增殖。该细胞系的成功构建, 进一步确证了NIH3T3-CD40L作为饲 养细胞可以促进体外低密度B-LCLs的增殖及提高EBV转化B淋巴细胞的效率, 优化了体外B 细胞 培养及EBV转化B淋巴细胞的流程, 也为单克隆抗体的制备奠定了基础。 关键词 NIH3T3-CD40L; 饲养细胞; B-LCLs Construction of NIH3T3-CD40L Stable Cell Lines and Its Application in EBV Transformation of B Lymphocytes Liu Xi, Zhao Ye, Xu Chongfeng, Duan Ziyuan* (Genetic Resource Research Center, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China) Abstract Stably expressing CD40L NIH3T3 cell lines were constructed and its application as feeder cells to culture and establish B lymphoblastoid cell lines (B-LCLs) in vitro were evaluated. pCMV6-CD40L was transfected into NIH3T3 cell lines through lipofectamine method, followed NIH3T3 that express CD40L were screened by G418. Positive clones were confirmed at RNA and protein level, the NIH3T3-CD40L cell lines were used as feeder cells in culturing B-LCLs and in the process of EBV transformation. The results showed that NIH3T3-CD40L cell lines increased the survival rate and promote the proliferation of B-LCLs in low densi