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负性共刺激分子B7-H4赋予肾癌细胞对化疗药物的耐药性研究

李金美, 宋慧, 练启慧, 陈敏吉, 章良, 谢炜

李金美, 宋慧, 练启慧, 陈敏吉, 章良, 谢炜. 负性共刺激分子B7-H4赋予肾癌细胞对化疗药物的耐药性研究[J]. 中国药科大学学报, 2013, 44(6): 553-558. DOI: 10.11665/j.issn.1000-5048.20130613
引用本文: 李金美, 宋慧, 练启慧, 陈敏吉, 章良, 谢炜. 负性共刺激分子B7-H4赋予肾癌细胞对化疗药物的耐药性研究[J]. 中国药科大学学报, 2013, 44(6): 553-558. DOI: 10.11665/j.issn.1000-5048.20130613
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LI Jinmei, SONG Hui, LIAN Qihui, CHEN Minji, ZHANG Liang, XIE Wei. Chemoresistance of negative costimulatory molecule B7-H4 to 786-O cells[J]. Journal of China Pharmaceutical University, 2013, 44(6): 553-558. DOI: 10.11665/j.issn.1000-5048.20130613
Citation: LI Jinmei, SONG Hui, LIAN Qihui, CHEN Minji, ZHANG Liang, XIE Wei. Chemoresistance of negative costimulatory molecule B7-H4 to 786-O cells[J]. Journal of China Pharmaceutical University, 2013, 44(6): 553-558. DOI: 10.11665/j.issn.1000-5048.20130613
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负性共刺激分子B7-H4赋予肾癌细胞对化疗药物的耐药性研究

  • 1.

    苏州大学医学部药学院

  • 2.

    苏州大学医学部基础医学与生命科学学院免疫学系

基金项目: 国家自然科学基金资助项目(No.30901361,No.31370872)

Chemoresistance of negative costimulatory molecule B7-H4 to 786-O cells

摘要

    摘要
  • 摘要: 通过RT-PCR方法克隆出人野生型负性共刺激分子B7-H4基因，将目的片段双酶切(EcoR Ⅰ和BamH Ⅰ)后，与pIRES2-EGFP真核表达载体连接，构建重组真核表达载体pIRES2-EGFP-B7-H4-WT并进行鉴定；然后用定点突变法构建B7-H4核定位序列突变体基因，再构建重组真核表达载体pIRES2-EGFP-B7-H4-NLS-MT；脂质体转染法将重组载体导入786-O细胞，CCK8法研究转基因细胞株的增殖率及对化疗药物5-氟尿嘧啶、阿霉素和顺铂的耐药性。结果显示:B7-H4野生型转基因细胞株相较于空白质粒组，可有效促进786-O细胞增殖，并赋予786-O细胞对3种化疗药物的耐药性。与空白质粒组相比，对5-氟尿嘧啶的耐药倍数为2.06，对阿霉素的耐药倍数为1.81，对顺铂的耐药倍数为1.72。机制研究显示B7-H4野生型可赋予肿瘤细胞抗凋亡作用，1.25 μg/mL 5-氟尿嘧啶引起B7-H4 WT/786-O的细胞的凋亡率是20.2%，而Mock/786-O和B7-H4 NLS/MT/786-O细胞的凋亡率分别是41.1%和35.1%。说明B7-H4可能通过调控细胞凋亡赋予肿瘤细胞多药耐药性。当B7-H4核定位序列被突变后，这些功能都消失，说明B7-H4促肿瘤细胞增殖、赋予其多药耐药性的功能与其核定位序列密切相关。
    Abstract: Human B7-H4 wild type gene was amplified by RT-PCR, digested with restriction endonuclease EcoR I and BamH I, and inserted into eukaryotic expression vector pIRES2-EGFP to construct recombinant eukaryotic expression vector pIRES2-EGFP-B7-H4-WT. The nuclear localization sequence(NLS)mutation type of B7-H4 was obtained by site-directed mutagenesis. The mutation type was then ligated with the vector pIRES2-EGFP and the recombinant eukaryotic expression vector pIRES2-EGFP-B7-H4-NLS-MT was constructed. The recombinant plasmids were transfected into 786-O cell line using Lipofectamine 2000 and three transgenic cells were constructed: Mock/786-O, B7-H4 WT/786-O and B7-H4 NLS MT/786-O. The cells proliferation and the cytotoxicity of chemotherapy drugs were assayed by CCK8 kit. The apoptosis of cells treated by 5-fluorouracil was assayed by Annexin V-PI/7ADD staining which detected by flowcytometry. Results showed that B7-H4 WT could effectively promote cell proliferation. When cells treated by various concentrations of 5-fluorouracil, doxorubicin and cisplatin B7-H4 WT/786-O cells were most resistant to drugs when compared with Mock/786-O and B7-H4 NLS MT/786-O, which suggested that B7-H4 wild type could confer chemoresistance to 786-O cells while B7-H4 NLS MT/786-O could not. The resistance index of B7-H4 WT/786-O cells to 5-5-fluorouracil was 2. 06, to doxorubicin was 1. 81 and to cisplatin was 1. 72. Flowcytometric analysis showed that when cells were treated with 1. 25 μg/mL 5-fluorouracil, the apoptosis of B7-H4 MT/786-O cells was lowest(20. 2%)when compared with Mock/786-O(41. 1%)and B7-H4 NLS MT/786-O(35. 1%). It showed that B7-H4 WT could confer multidrug resistance to 786-O cells through regulating cell apoptosis. However B7-H4 NLS MT could not act as an anti-apoptotic molecule. These results demonstrate that the NLS play an important role in the proliferation and chemoresistance promotion effect of B7-H4.
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    • 参考文献(22)
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  • 刊出日期:  2013-12-24

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