东方田鼠皮肤成纤维永生化细胞的建立

Establishment of an immortalized Microtus fortis skin fibroblast cell line

为建立东方田鼠皮肤成纤维细胞(MfSF)系,本研究采用脂质体基因转染法将含有SV40T基因的pSV3neo重组质粒导入原代MfSF中,通过G418筛选,阳性克隆扩大培养,建立永生化M fSF系。实验结果表明,阳性细胞克隆在G418筛选第三周后出现,经扩大培养已稳定传代达42代,而且生长状态良好,PCR及RT-PCR检测结果表明,SV40 T基因已整合到M fSF中并稳定表达。本研究利用SV40T基因导入制备了永生化M fSF细胞系,为动物间成纤维细胞比较研究及相关病原的研究提供了敏感细胞系。

In order to establish an immortalized Microtus fortis skin fibroblast （MfSF） line, a mammalian expression recombinant plasmid of pSV3neo containing the SV40 large T antigen gene was used to transfect into the primary MfSF cells using LipofectarnineTM 2000. The positive colonies were appeared after 3 weeks screened by G418, and expanded into immortalized cell lines which passed stably for 42 passages. In addition, the integration of the large T antigen gene into the genome of the immortalized MfSF and the mRNA of SV40 large T antigen in the expanded cells were detected by PCR and RT-PCR, respectively, which substantially proved the immortalized MfSF line was established. The established MfSF line provided a basis for comparative study of fibroblasts fi＇om different animals and the etiological studies in vitro.