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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

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

  • 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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