稳定表达T-钙黏蛋白的黑色素瘤顺铂耐药细胞株的建立及其生物学特性  

Establishment of cisplatin-resistant melanoma cell line with stable expression of T-cadherin and its biological characteristics

作　　者：陆海涛[1] 郭健[1] 何磊[1] 刘利君[1] 段昕所[1] LU Haitao;GUO Jian;HE Lei;LIU Lijun;DUAN Xinsuo(Department of Dermatology,Affiliated Hospital of Chengde Medical College,Chengde Hebei 067000,China)

机构地区：[1]承德医学院附属医院皮肤科

出　　处：《中南大学学报（医学版）》2019年第11期1214-1221,共8页Journal of Central South University ：Medical Science

摘　　要：目的:建立稳定表达T-钙黏蛋白(T-cadherin)基因的顺铂(cisplatin,CDDP)耐药黑色素瘤B16F10细胞株(CDDP-R B16F10),并研究其生物学特性。方法:采用大剂量冲击和逐步增加剂量相结合的方法诱导CDDP-R B16F10细胞株。采用四甲基偶氮唑盐[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide,MTT]法检测CDDP-R B16F10细胞株的增殖能力,并观察CDDP-R B16F10细胞株对CDDP和紫杉醇的敏感性。将T-cadherin互补cDNA插入增强绿色荧光蛋白质粒(enhanced green fluorescent protein plasmid,pEGFP)-N1载体中,获得编码人T-cadherin的质粒载体pEGFP-N1-T-cadherin,再将pEGFP-N1-T-cadherin转染CDDP-R B16F10细胞。分别采用反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)、蛋白质印迹法和免疫组织化学SP法检测转染后耐药细胞株T-cadherin的mRNA及蛋白质表达情况。采用MTT法检测T-cadherin联合CDDP对CDDP-R B16F10细胞株增殖的影响,并观察转染T-cadherin后CDDP-R B16F10细胞株对紫杉醇的敏感性。结果:成功建立耐CDDP的CDDP-R B16F10细胞株。该细胞株同B16F10细胞株比较,其增殖能力差异无统计学意义(P>0.05)。CDDP-R B16F10细胞株和B16F10细胞株对CDDP的半数抑制浓度(half maximal inhibitory concentration,IC50)分别为268.706和19.748 mg/L,耐药指数为13.61。CDDP-R B16F10细胞株和B16F10细胞株对紫杉醇的IC50分别为11.415和7.799 mg/L,耐药指数为1.46。重组真核表达载体pEGFPN1-T-cadherin构建成功。RT-PCR,蛋白质印迹法和免疫组织化学SP法检测结果显示:CDDP-R B16F10可转录和表达T-cadherin。MTT法显示T-cadherin联合CDDP可抑制CDDP-R B16F10细胞株的增殖(P<0.05),析因分析显示T-cadherin与CDDP联合对CDDP-R B16F10细胞株增殖的抑制有交互效应(P<0.05)。T-cadherin联合紫杉醇可抑制CDDP-R B16F10细胞株的增殖(P<0.05),析因分析显示T-cadherin与紫杉醇联合对CDDP-R B16F10细胞株增殖的抑制无交互效应(P>0.05)。结论:成�Objective:To establish cisplatin(CDDP)-resistant melanoma B16F10(CDDP-R B16F10)cell line with stable expression of T-cadherin,and to study its biological characteristics.Methods:CDDP-R B16F10 cell line was exposure to high and gradually increased dose of CDDP.3-(4.5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)was used to test the proliferation of CDDP-R B16F10 cell line,and the sensitivity of CDDP-R B16F10 cell line to CDDP and paclitaxel was examined.The pEGFP-N1-T-cadherin,a plasmid vector encoding human T-cadherin,was generated by inserting T-cadherin cDNA into a pEGFP-N1 vector.The pEGFPN1-T-cadherin was transfected into CDDP-R B16F10 cell line.The expression of T-cadherin mRNA and protein were measured by reverse transcription polymerase chain reaction(RT-PCR),Western blotting and immunohistochemistry SP method,respectively.The effect of T-cadherin combined with CDDP on proliferation of CDDP-R B16F10 cell line was determined by MTT assay.The sensitivity of CDDP-R B16F10 cell line with stably transfected T-cadherin to paclitaxel was examined by MTT assay.Results:The CDDP-R B16F10 cell line was established successfully.There was no difference in proliferation between the CDDP-R B16F10 cell line and B16F10 cell line(P>0.05).The IC50 of CDDP-R B16F10 cell line and B16F10 cell line to CDDP were 268.706 and 19.748 mg/L,respectively,and the resistance index was 13.61.The IC50 of CDDP-R B16F10 cell line and B16F10 cells to paclitaxel were 11.415 and 7.799 mg/L,respectively,and the resistance index was 1.46.The expression vector pEGFP-N1-T-cadherin was constructed successfully.RT-PCR,Western blotting and immunohistochemistry SP method showed that T-cadherin could be transcribed and expressed.MTT assay showed that T-cadherin combined with CDDP could inhibit the proliferation of CDDP-R B16F10 cell line(P<0.05).Factorial analysis showed that there was interaction between T-cadherin and CDDP in inhibiting the proliferation of CDDP-R B16F10 cell line(P<0.05).T-cadherin combined with paclitaxel could inhibit

关 键 词：T-钙黏蛋白 顺铂 黑色素瘤 耐药性 

分 类 号：R73[医药卫生—肿瘤]