摘要
目的探讨巨噬细胞移动抑制因子(Macrophage migration inhibitory factor,MIF)重组质粒转染人宫颈癌SiHa细胞后MIF的表达及其对CyclinD1表达的影响。方法构建并鉴定重组真核表达质粒pEGFP-N1-MIF,转染人宫颈癌SiHa细胞;ELISA法检测细胞上清液中MIF的表达;real-time PCR及免疫细胞化学法检测细胞中MIF、CyclinD1 mRNA及蛋白的表达水平;MTT法及Boyden小室法分别检测细胞增殖和体外迁移能力。结果重组质粒pEGFP-N1-MIF成功构建并转染至SiHa细胞;ELISA证实转染pEGFP-N1-MIF的实验组细胞上清液中MIF表达均高于各对照组(P<0.01);real-time PCR及免疫细胞化学法证实实验组细胞中MIF、CyclinD1 mRNA和蛋白的表达水平均高于各对照组(P<0.01);MTT法和Boyden小室法检测到转染pEGFP-N1-MIF后细胞体外增殖、迁移能力明显高于各对照组(P<0.01)。结论转染重组质粒pEGFP-N1-MIF后SiHa细胞中MIF表达水平增高,MIF过表达可增强SiHa细胞体外增殖、迁移能力,并上调CyclinD1的表达。
Objective To investigate the expression of macrophage migration inhibitory factor（MIF） in human cervical cancer cells SiHa transfected MIF recombinant plasmid and its influence on CyclinD1 expression. Methods Recombinant eukaryotic expression plasmid pEGFP-N1-MIF was constructed and identified, then transfected into human cervical cancer cells SiHa using a liposome approach. The expression of MIF in supernatant fluid was detected by ELISA. The changes of MIF, CyclinD1 mRNA and protein expression were detected by real-time PCR and immunocytochemistry respectively. MTT assay and Boyden small chamber were performed to examine the changes of cell proliferation and migration in vitro respectively. Results The recombinant plasmid pEGFP-N1-MIF was constructed successfully and transfected into SiHa ceils. The expression level of MIF in supematant fluid of the experimental group was higher than that in negative control group and blank control group （P〈0.01）. After transfection, the MIF, CyclinD1 mRNA and protein expression level in experimental group were significantly higher than that in negative control group and blank control group （P〈0.01）. In comparison with negative control group and blank control group, the proliferation and migration ability in vitro of experimental group was significantly higher（P〈0.01）. Conclusion MIF expression level in SiHa cells is increased after recombinant plasmid transfection. The over-expression of MIF gene can significantly promote proliferation and migration ability of SiHa cells in vitro, and up-regulate the expression of CyclinD 1.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2013年第10期949-953,共5页
Cancer Research on Prevention and Treatment
基金
山西省自然科学基金资助项目(2010011047-3)
