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蛋白表达抑制稳定株MDA—MB-231-Tudor—SNI的筛选和鉴定

ISSN号：1672-8009
期刊名称：《医学分子生物学杂志》
时间：0
分类：R349.64[医药卫生—基础医学]
作者机构：[1]天津医科大学免疫学教研室,天津市300070, [2]国家教育部免疫微环境与疫病重点实验室,天津市300070, [3]天津市细胞与分子免疫学重点实验室,天津市300070
相关基金：国家高新技术研究发展计划（863计划）（No.2007AA022115）,国家自然科学基金（No.30670441,30970562,30670802,30811130394,30911130165,30970582）,国家重点基础研究发展规划（973计划）（No.2009CB918903）,天津市科委国际合作项目（N0.07JCZDJC07300）,国家自然科学重大研究计划培育项目（No.90919032）,国家教育部高等学校博士学科点专项科研基金（No.20091202110001）,天津市教委重点项目（No.2008ZD01）

作者：王保亚[1,2,3], 刘欣[1,2,3], 高星杰[1,2,3], 朱梦瑜[1,2,3], 杨振霞[1,2,3], 赵秀娟[1,2,3], 杨洁[1,2,3]

关键词：人类Tudor—SN蛋白, RNA干扰, 细胞筛选, 稳定株, human Tudor-SN protein, RNAi, cell screening, stable transfectant

中文摘要：

目的采用针对人类Tudor—SN基因的RNA干扰（RNAi）技术建立Tudor—SN表达抑制的乳腺癌MDA—MB-231稳定细胞株。方法利用脂质体转染方法将pGenesil—shRNA—hTudor—SNI质粒转染进入MDA—MB-231细胞，经G418筛选出稳定株，通过RT—PCR检测Tudor—SN的mRNA水平，再以Western印迹技术鉴定Tudor—SN蛋白表达情况并计算蛋白表达抑制率。结果与对照组相比，以G418筛选的MDA—MB-231-Tudor—SNI稳定株Tudor—SN的mRNA水平降低（P〈0．01，n=3），蛋白表达水平明显下调（P〈0．01，n=3），蛋白表达抑制率达78．12％。结论成功筛选并鉴定人类Tudor—SN蛋白表达抑制MDA—MB-231稳定株，有助于深入研究Tudor—SN蛋白在乳腺癌中的作用。

英文摘要：

Objective To transfect the constructed plasmid expressing down-regulation of Hu- man Tudor-SN Gene into MDA-MB-231 cells and screen the cell lines of stable interference human Tudor-SN gene. Methods Recombinant vector pGenesil-shRNA-hTudor-SNI was transfected into MDA-MB-231 cells using Lipofectamine-2000. The cell strains for stable and down-expression of Tudor-SN protein with the medium containing G418 sulfate were selected. The expression level of Tudor-SN was then detected by RT-PCR and western blotting assay. Results The hTudor-SN mRNA and protein expression levels in stable transfectant screened with G418 were down-regulated as com- pared to those in negative control group （P 〈0. 01, n = 3） . The inhibition rate of Tudor-SN protein expression was up to 78.12%. Conclusion The stable MDA-MB-231-Tudor-SNI cell lines were screened and examined successfully. The results are useful for research on the function of Tudor-SN protein on breast cancer.

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