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

作　　者：李金美[1] 宋慧[1] 练启慧 陈敏吉 章良[1] 谢炜[2] 

机构地区：[1]苏州大学医学部药学院,苏州215123 [2]苏州大学医学部基础医学与生命科学学院免疫学系,苏州215123

出　　处：《中国药科大学学报》2013年第6期553-558,共6页Journal of China Pharmaceutical University

基　　金：国家自然科学基金资助项目(No.30901361;No.31370872)~~

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(EcoRⅠ和BamHⅠ)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,1.25μg/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。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 786-0 cell line using Lipofectamine 2000 and three transgenic cells were construc- ted： 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 cispla- tin 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-Ocells 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/mL5-fluorouracil, the apoptosis of B7-H4 MT/786-O cells was lowest （20. 2%） when compared with Mock/786-O （41.1%） and BT-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.

关 键 词：B7-H4 肾癌细胞 5-氟尿嘧啶 核定位序列 耐药性 

分 类 号：R737.11[医药卫生—肿瘤]