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目的:利用慢病毒载体短发夹RNA(shRNA)介导人乳腺癌细胞ERβ基因沉默,筛选鉴定,并建立ERβ基因稳定下调的乳腺癌细胞株。方法:将靶向沉默ERβ基因的shRNA慢病毒颗粒感染人乳腺癌细胞株T47D和MCF-7,以未感染及空载体慢病毒感染的T47D和MCF-7细胞分别作为空白对照和阴性对照。先以慢病毒瞬转48 h,通过蛋白免疫印迹法(western-blot)进行蛋白水平检测筛选出干扰效果最好的两组,然后继续经浓度为1 mg/L的嘌呤霉素连续筛选4周,采用RT-PCR和western-blot方法,分别对ERβ在mRNA和蛋白水平上的沉默效果进行鉴定。结果:慢病毒感染乳腺癌细胞后,与阴性对照组相比,实验组ERβmRNA和蛋白表达量均明显下降(P〈0.05):其中T47D细胞株shRNA3326、3327两实验组下调效果最明显,ERβmRNA水平和蛋白水平分别达到(61.12±3.66)%、(76.47±3.16)%和(60.83±3.07)%、(53.31±3.00)%;MCF-7细胞株shRNA3325、3326两实验组下调效果最显著,ERβmRNA水平和蛋白水平下调率分别为(62.42±0.07)%、(42.49±1.96)%和(83.69±5.07)%、(73.16±13.21)%。而阴性对照与空白对照组相比无显著性差异,无统计学意义(P>0.05)。结论:成功筛选并建立了ERβ基因稳定下调的两株乳腺癌细胞系T47D和MCF-7,从而为后续探究改变ERβ表达水平在乳腺癌发生发展及在乳腺癌内分泌治疗效果中的作用提供有用的细胞研究模型。
Objective: To use lentiviral vector short hairpin RNA (shRNA) to mediate ERβ gene silencing in human breast cancer cells, screen and identify, and establish a stable down-regulation of ERβ gene in breast cancer cell lines. METHODS: Human breast cancer cell lines T47D and MCF-7 were infected with shRNA lentiviral particles targeted to silence the ERβ gene. T47D and MCF-7 cells infected with uninfected and empty vector lentivirus were used as blank and negative controls, respectively. First transiently 48 h with lentivirus, protein levels were detected by Western-blot for the two groups with the best interference, and then continued for 4 weeks with puromycin at a concentration of 1 mg/L. RT-PCR and western-blot methods were used to identify the silencing effects of ERβ on mRNA and protein levels, respectively. Results: Compared with the negative control group, the expression of ERβ mRNA and protein in the experimental group was significantly decreased after the Lentivirus infection of breast cancer cells (P<0.05): The T47D cell lines shRNA3326 and 3327 had the most obvious down-regulation effect and the ERβ mRNA level The levels of protein and protein were (61.12±3.66)%, (76.47±3.16)% and (60.83±3.07)%, and (53.31±3.00)%, respectively; the MCF-7 cell lines shRNA3325 and 3326 had the most significant down-regulation effect, ERβmRNA. The rates of down-regulation of levels and protein levels were (62.42±0.07)%, (42.49±1.96)%, and (83.69±5.07)%, (73.16±13.21)%, respectively. There was no significant difference between the negative control and the blank control group (P>0.05). Conclusion: Two breast cancer cell lines T47D and MCF-7 with stable down-regulation of ERβ gene were successfully screened and established, which could be useful for the subsequent exploration of the role of changing ERβ expression in the development of breast cancer and its role in endocrine treatment of breast cancer. Cell research model.