稳定表达IL-8基因宫颈癌SiHa细胞株的建立及鉴定

Establishment and identification of SiHa cell line stably and highly expressing IL-8 gene

目的建立高效、稳定表达IL-8基因的人宫颈癌pcDNA3.1-IL-8-SiHa细胞株。方法利用pcDNA3.1(+)真核表达载体构建pcDNA3.1/IL-8表达载体,利用基因克隆技术将IL-8cDNA基因片段通过脂质体转染入人宫颈鳞癌SiHa细胞中,经G418筛选获得稳定表达的细胞克隆,建立了高表达IL-8的pcDNA3.1-IL-8-SiHa宫颈癌细胞株,转染阳性细胞连续传代培养,传8代后用RT-PCR,ELISA,免疫细胞化学法进一步鉴定细胞中IL-8的表达。结果成功建立了持续高表达IL-8的pcDNA3.1-IL-8-SiHa细胞株,并在细胞中检测到IL-8mRNA及蛋白的高表达。结论高效表达IL-8基因的SiHa细胞株的建立有助于研究IL-8基因在宫颈癌发生发展中的作用。

Objective To establish a cell line pcDNA3.1-IL-8-SiHa efficiently and stably expressing IL-8 gene.Methods The pcDNA3.1（＋）expression vector was used to construct the expression vector of the pcDNA3.1/IL-8.The two vectors were transferred to human cervical cancer SiHa cell line through cloning technology of lipofectin,respectively.By G418 the stably expressing cell clones were screened for establishing the pcDNA3.1-IL-8-SiHa cervical cancer cell lines highly expressing IL-8 gene.The transfection-positive cells were continuously subcultured,after 8th passage,the expression of IL-8 was detected by RT-PCR,ELISA,immunocytochemical staining at mRNAand protein levels.Results pcDNA3.1-IL-8-SiHa cell line stably expressing IL-8 gene was constructed successfully,and IL-8 mRNA and protein expressed highly in the cells.Conclusion The establishment of the cell line stably and highly expressing IL-8 gene can help to study the roles of IL-8 gene in the development of human cervical squamous carcinoma cancer.