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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。

英文摘要:

Objective: To establish a stable ERβ gene silenced cell line of human breast cancer cells by using short hairpin RNA (shRNA) lentiviral vector. Methods: The recombinant lentivirus and negative control lentivirus were used to infect MCF-7 and T47D cells. The non-transfected and empty vectorMCF-7 and T47D cells were respectively taken as the blank control and negative control. After infected for 48 h, two groups with best interference effect were screened out by Western blot method. Then the two groups were screened by purimycin (1 mg/L) for 4w, and then the ERβ mRNA and protein level were detected by RT-PCR and Western blotting. Results: The expression of ERβ in MCF-7 and T47D cells was significantly decreased at both mRNA and protein level in experimental group, as compared with negative control group (P<0.05). The T47D-ERβ shRNA-3326 and T47D-ERβ shRNA-3327 played a significant role in reducing respectively mRNA level to (61.12±3.66)% and (60.83±3.07) %, protein level to (76.47±3.16)% and (53.31±3.00)% (P<0. 05); And MCF-7-ERβ shRNA-3325 and MCF-7-ERβ shRNA-3326 also played a significant role in reducing mRNA level to (62.42± 0.07)% and (83.69±5.07)%, protein level to (42.49±1.96)% and (73.16±13.21)% respectively (P<0.05); the silence effect showed no significant difference between the non-transfected group and negative control group (P>0.05). Conclusions: The successful establishment of a stable and effectively reduced ERβ gene silenced cell lines of two breast cancer cells T47D and MCF-7 provides a powerful and useful cell research model for further study on the role of ERβ gene in the progression and endocrine therapy of breast cancer.

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