| 李江鹏1 赵华栋1 张健2 李燕2 何显力1.慢病毒介导短发夹ShRNA 沉默ERβ 乳腺癌细胞株的建立[J].,2012,12(16):3029-3032 |
| 慢病毒介导短发夹ShRNA 沉默ERβ 乳腺癌细胞株的建立 |
| Lentiviral Vector-Mediated Short Hairpin RNA Silences ERβ Gene inHuman Breast Cancer Cells Line |
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| DOI： |
| 中文关键词: 雌激素受体β(ERβ) 乳腺癌细胞株 短发夹状RNA 慢病毒 感染 |
| 英文关键词: ERβ Breast cancer cells line Short hairpin RNA(shRNA) Lentivirus Transfect |
| 基金项目:国家自然科学基金(30972930)；第四军医大学唐都医院精英人才培育资助计划 |
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| 中文摘要: |
| 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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