PC-1基因表达诱导NIH3T3细胞恶性转化

Malignant transformation of NIH3T3 cells induced by ectopic expression of PC-1 gene

目的 通过建立稳定表达外源PC -1基因的小鼠成纤维细胞株,初步探讨PC- 1基因表达对肿瘤发生、发展的影响。方法 通过脂质体介导的方法,将真核表达载体pcDNA3 1( ) /myc his pc 1稳定转染NIH3T3细胞,之后利用PCR、逆转录PCR(RT PCR)技术,确定外源PC -1基因在靶细胞染色体上的整合及在转录水平的表达。通过细胞形态学分析、MTT实验、细胞周期分析、软琼脂集落形成和裸鼠成瘤实验,观察PC -1基因表达对NIH3T3生物学特性的影响。结果 建立了稳定转染PC- 1基因的NIH3T3细胞株。PC -1基因表达的小鼠成纤维细胞NIH3T3生长速度加快,在软琼脂上生长并形成集落,接种裸鼠后可成瘤 (6 /6)。结论 PC- 1基因在NIH3T3细胞中稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。

Objective To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene. Methods Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression.Results NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice.Conclusion Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.