山东医药2016，Vol.56Issue(16)：19-22,4.DOI:10.3969/j.issn.1002-266X.2016.16.006
慢病毒介导干扰 LSD1基因的卵巢癌稳转细胞株的构建及鉴定
Construction and identification of stably transfected ovarian cancer cell lines with lentiviral RNA interference-mediated down-regulation of LSD1 gene
摘要
Abstract
Objective To construct the stably transfected ovarian cancer cell lines SKOV 3 with lentiviral RNA inter-ference-mediated down-regulation of lysine-specific demethylase 1 (LSD1) gene and to provide basis for the in-depth study of taking LSD1 gene as a target in the epigenetics anti-tumor treatment.Methods The plasmids was amplified and extrac-ted, and the UV spectrophotometer was used to detect their purity , recording A260/A280 .PLKO-TET-On plasmids were transfected into 293T cells via Lipofectamine 2000, The transfection of 293T cells was observed under an inverted fluores-cence microscope .Collecting virus supernatants which were infected by ovarian cancer cells SKOV 3 ( observation group ) and the vector PLKO was used as the negative control ( control group ) .The stably transfected cell lines were selected .The cell lines were induced by doxycycline (0, 1, 10, 100 and 1 000 ng/mL) in the observation group , and then they were both induced by 100 ng/mL doxycycline, the expression of LSD1, H3K4me1 and H3K4me2 in the stably transfected cell lines was detected by Western blotting .Results Ultraviolet spectrophotometer showed that the target plasmid A 260/A280 was around 2.0 with high purity, THM plasmid-transfected 293T cells appeared green fluorescence , which was eukaryotic expression vector of GFP green fluorescent protein , suggesting that transfection was successful .The protein expression level of LSD1 was decreased and its main substration H3K4me1, H3K4me2 expression was increased with the concentration gra-dient of doxycycline.There were statistically significant differences between multiple concentrations (all P<0.01).The relative expression of LSD 1 protein of the observation group induced by 100 ng/mL doxycycline was significantly decreased but the expression of H3K4me1, H3K4me2 was increased as compared with that in the group without doxycycline induction and the control group after the induction of doxycycline (all P<0.01).Conclusion The stably transfected ovarian cancer cell lines SKOV3 with lentiviral RNA interference-mediated down-regulation of LSD1 gene are constructed successfully and identified with concentration gradient of doxycycline induction ;this cell line can be used in the epigenetics anti-tumor treat-ment of taking LSD1 as a target.关键词
赖氨酸特异性去甲基化酶1/RNA干扰/人卵巢癌SKOV3细胞/稳转细胞株Key words
lysine-specific demethylase 1/RNA interference/human ovarian cancer SKOV 3 cells/stably transfected cells分类
医药卫生引用本文复制引用
万晓蕾,赖文升,邵根宝,李袁霞,刘秀雯,金洁,吴朝阳..慢病毒介导干扰 LSD1基因的卵巢癌稳转细胞株的构建及鉴定[J].山东医药,2016,56(16):19-22,4.基金项目
国家自然科学基金资助项目（81170573）。 （）
