遗传 ›› 2012, Vol. 34 ›› Issue (6): 705-710.doi: 10.3724/SP.J.1005.2012.00705

• 研究报告 • 上一篇    下一篇

mbr-FPGS高效表达质粒增强Jurkat细胞对甲氨蝶呤的敏感性

胡文佳1, 孙玉洁1,2   

  1. 1. 南京医科大学细胞生物学系, 南京210029 2. 江苏省人类基因组重点实验室, 南京210029
  • 收稿日期:2012-01-08 修回日期:2012-03-29 出版日期:2012-06-20 发布日期:2012-06-25
  • 通讯作者: 孙玉洁 E-mail:yujiesun@njmu.edu.cn
  • 基金资助:

    国家自然科学基金项目(编号:90919051)资助

The mbr-FPGS efficient expression plasmid enhances the sensitivity of Jurkat cells to methotrexate

HU Wen-Jia1, SUN Yu-Jie1, 2   

  1. 1. Department of Cell Biology, Nanjing Medical University, Nanjing 210029, China 2. Key Laboratory of Human Functional Genomics of Jiangsu Province, Nanjing 210029, China
  • Received:2012-01-08 Revised:2012-03-29 Online:2012-06-20 Published:2012-06-25

摘要: 叶酰多聚谷氨酸盐合成酶(Folylpolyglutamate synthetase, FPGS)是将化疗药物甲氨蝶呤(Methotrexate, MTX)转化成甲氨蝶呤多聚谷氨酸盐(MTXPG)的关键酶, 其表达水平直接影响肿瘤细胞对MTX敏感性。与B细胞急性淋巴细胞白血病(B-ALL)相比, T细胞急性淋巴细胞白血病(T-ALL)细胞中FPGS表达水平低, 因此对MTX不敏感。本实验室前期研究证实, 位于BCL2基因3′-UTR区的一段长279 bp的 DNA序列mbr具有显著的增强子效应。文章构建了含有mbr增强子样序列的FPGS表达质粒, 用其转染Jurkat细胞后, 分别以Western blotting和MTT法检测FPGS表达水平及MTX对肿瘤细胞的抑制率。结果表明mbr能够显著提高FPGS表达质粒的表达水平, 并有效增强Jurkat细胞对MTX的敏感性。这一结果为将基础研究结果应用于临床、提高MTX对T-ALL细胞的化疗疗效提供了新的思路。

关键词: FPGS基因, mbr, 过表达, MTX敏感性

Abstract: Folylpolyglutamate synthetase (FPGS) is the key enzyme that converts chemotherapy drug Methotrexate (MTX) into MTXPG. The expression level of FPGS directly influences MTX-sensitivity of tumor cells. Compared with B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL) cells express a lower level of FPGS, which results in insensitivity of the cells to MTX. Our previous work has demonstrated that 279 bp mbr element located within the 3′-UTR of the BCL2 gene possesses enhancer function. In this study, FPGS expression plasmid containing mbr element at the 5′ upstream of the gene was constructed and transfected into Jurkat cells to sensitize the cells to MTX. Western blotting and MTT assay were applied to detect the FPGS expression level and suppression rate of the cells treated by MTX, respectively. We found that the mbr enhanced the expression of FPGS significantly and increased sensitivity of Jurkat cells to MTX efficiently, while FPGS expression plasmid without mbr element had less effect. Our data provides a new clue for the clinical application of mbr regulatory element and may contribute to improvement of MTX treatment in T-ALL.

Key words: FPGS, mbr element, overexpression, MTX-sensitivity