[1]李继强　杨吉安　邵灵敏　吴庭枫　刘宝辉　陈谦学.稳定低表达BAG3的胶质母细胞瘤U87细胞株的构建及鉴定[J].中国临床神经外科杂志,2015,(06):353-355.[doi:10.13798/j.issn.1009-153X.2015.06.011]
　LI Ji-qiang,YANG Ji-an,SHAO Ling-min,et al.Construction and identification of U87 glioblastoma cell strain with a stable low expression of BAG3[J].,2015,(06):353-355.[doi:10.13798/j.issn.1009-153X.2015.06.011]
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稳定低表达BAG3的胶质母细胞瘤U87细胞株的构建及鉴定()
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《中国临床神经外科杂志》[ISSN:1009-153X/CN:42-1603/TN]

卷:
期数:
2015年06期
页码:
353-355
栏目:
论著
出版日期:
2015-06-30

文章信息/Info

Title:
Construction and identification of U87 glioblastoma cell strain with a stable low expression of BAG3
文章编号:
1009-153X（2015）06-0353-04
作者:
李继强　杨吉安　邵灵敏　吴庭枫　刘宝辉　陈谦学
430060 武汉，武汉大学人民医院神经外科
通讯作者：陈谦学，E-mail：chenqx666@sohu.com
Author(s):
LI Ji-qiang;  YANG Ji-an;  SHAO Ling-min;  WU Ting-feng;  LIU Bao-hui;  CHEN Qian-xue.
Department of Neurosurgery, Renmin Hospital, Wuhan University, Wuhan 430060, China
关键词:
胶质母细胞瘤; U87细胞株; BAG3; RNA干扰
Keywords:
Glioblastoma;  U87 cell strain;  BAG3;  RNA interference
分类号:
R 739.41; Q 789
DOI:
10.13798/j.issn.1009-153X.2015.06.011
文献标志码:
A
摘要:
目的 探讨构建稳定低表达BAG3的胶质母细胞瘤U87细胞株的方法。方法 利用RNA干扰技术提取pEGFP-BAG3- shRNA质粒后采用ABI3130基因测序仪进行测序鉴定，将鉴定后的质粒分别转染到胶质母细胞瘤U87细胞株，利用嘌呤霉素筛选转染的U87细胞，通过RT-PCR、免疫印迹分析和免疫荧光染色等技术对转染后的U87细胞进行鉴定。结果 pEGFP-BAG3-shRNA质粒测序结果包含干 扰序列，筛选后的干扰组和对照组U87细胞转染效率分别为85%和93%，RT-PCR结果显示shBAG3质粒干扰组BAG3 mRNA表达量显著低于对照组（P<0.05）；免疫印迹分析及免疫荧光染色结果显示shBAG3质粒干扰组BAG3 蛋白表达量显著低于对照组（P<0.05）。结论 本研究所构建的胶质母细胞瘤U87细胞株能稳定低表达BAG3，可用于后续BAG3在胶质母细胞瘤中的生物学作用及相关信号通路的研究。
Abstract:
Objective To construct and identify glioblastoma U87 cell strain with a stable low expression of BAG3. Methods The plasmids of pEGFP-BAG3-shRNA were extracted by RNA interference technique and then were sequence by gene sequencing instrument ABI3130. The plasmids of pEGFP-BAG3-shRNA which met the sequence identical outcomes were transfected into U87 glioblastoma cell strain. The transfected U87 glioblastoma cell strains were sifted out by puromycin and then were identified by RT-PCR, western-blotting and immunofluorescence techniques. Results The sequencing outcomes showed that pEGFP-BAG3-shRNA plasmid contained Objective gene interfering sequence. The percentage of tranfected U87 glioblastoma in shBAG3 group and shCtrl group were 85% and 93% respectively. The levels of BAG3 mRNA and protein expressions were significantly lower in shBAG3 group than those in shCtrl group. Conclusion U87 glioblastoma cell strain constructed by the present study can stably express BAG3 at a low level and may be used for the further research of the role of BAG3 in the gliobalstoma and its associated signaling pathways.

参考文献/References:

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更新日期/Last Update: 2015-06-30