摘要
目的构建并鉴定慢病毒介导干扰赖氨酸特异性去甲基化酶1(LSD1)基因的卵巢癌稳转细胞株,为深入研究以LSD1为靶点的表观遗传学抗肿瘤治疗奠定基础。方法扩增并抽提所需质粒,紫外分光光度计检测质粒纯度,计算A260/A280。采用Lipofectamine 2000脂质体转染法将质粒转染入人肾上皮细胞293T,取转染THM质粒后的293T细胞,在倒置荧光显微镜下观察转染情况;分别取转染目的质粒和PLKO空载体质粒后的293T细胞,收集病毒上清液,感染人卵巢癌SKOV3细胞(分别记为观察组和对照组),筛选出稳转细胞株。观察组经浓度梯度(0、1、10、100、1 000 ng/m L)多西环素诱导,以及两组均经100 ng/m L多西环素诱导后,采用Western blotting法检测LSD1及其特异性反应底物H3K4me1、H3K4me2蛋白相对表达量。结果目的质粒A260/A280在2.0左右,说明纯度较高;THM质粒转染293T细胞呈绿色荧光,为真核载体表达的绿色荧光蛋白,表明成功转染。随着多西环素浓度的升高,观察组诱导后LSD1蛋白相对表达量均逐渐降低,H3K4me1、H3K4me2蛋白相对表达量逐渐升高,各浓度间比较有统计学差异(P均<0.01)。观察组经100 ng/m L多西环素诱导后LSD1蛋白相对表达量均明显低于同组诱导前及对照组诱导后,H3K4me1、H3K4me2蛋白相对表达量明显高于同组诱导前及对照组诱导后(P均<0.01)。结论成功构建慢病毒干扰下调LSD1基因表达的卵巢癌稳转细胞株,并经不同浓度多西环素诱导鉴定;该细胞株可用于以LSD1为靶点的表观遗传学抗肿瘤治疗研究。
Objective To construct the stably transfected ovarian cancer cell lines SKOV3 with lentiviral RNA interference-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 extracted,and the UV spectrophotometer was used to detect their purity,recording A260/ A280. PLKO-TET-On plasmids were transfected into 293 T cells via Lipofectamine 2000,The transfection of 293 T cells was observed under an inverted fluorescence microscope. Collecting virus supernatants which were infected by ovarian cancer cells SKOV3（ 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 / m L） in the observation group,and then they were both induced by 100 ng / m L 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 A260/ A280 was around 2. 0 with high purity,THM plasmid-transfected 293 T 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 gradient of doxycycline. There were statistically significant differences between multiple concentrations（ all P〈0. 01）. The relative expression of LSD1 protein of the observation group induced by 100 ng / m L 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
出处
《山东医药》
CAS
北大核心
2016年第16期19-22,共4页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81170573)
