人绒毛膜癌JAR/VP16多药耐药细胞株的建立及相关基因表达检测

Establishment of a multidrug resistant cell line JAR/VP16 of human choriocarcinoma and analyses of the expression of multidrug resistance-and apoptosis-associated genes

目的:建立人绒毛膜癌鬼臼乙叉甙(VP16)耐药细胞株,检测其生物学特性及耐药和凋亡相关基因的表达,探讨多药耐药性产生与凋亡基因改变之间的相关性。方法:体外采用VP16浓度梯度递增法诱导建立JAR/VP16耐药细胞株,终浓度药物维持培养4个月,MTT法测定多药耐药谱及耐药稳定性;倒置相差显微镜观察并结合HE染色探讨耐药细胞在光镜下的形态学变化,电镜观察细胞的超微结构;基因芯片检测耐药相关基因及凋亡相关基因的表达。结果:①历经14个月、70多代耐药诱导培养,成功构建人绒毛膜癌JAR细胞耐VP16的耐药细胞株,命名为J“AR/VP16耐药细胞株,”与JAR细胞相比有显著的形态学改变,其对VP16的耐药性能稳定,耐药指数(RI)为73.67;并且对紫杉醇(TAX)、甲氨蝶呤(MTX)和更生霉素(KSM)具有显著的交叉耐药性,耐药指数分别为26.18、18.67和14.73;对5-氟尿嘧啶(5-FU)和长春新碱(VCR)的耐受性有所增加(RI∶2.88;3.45);对阿霉素(ADM)则无明显的交叉耐药(RI∶1.39)。②基因芯片结果表明,JAR/VP16细胞MRP1、MRP2、MRP6、BCRP、LRP和Rb等耐药基因表达明显高于亲本细胞,并且Bcl-2、Bcl-x、MyD88、TRAF-4、TRAF-6、Trip等凋亡抑制基因表达上调,Bax、BimL、Blk、Caspase-7-、8、-13-、14以及Fas和FasL等凋亡促进基因表达下调。结论:①JAR/VP16细胞呈现多药耐药性且耐药性能稳定,为研究耐药机制及筛选药物提供了理想的实验模型。②JAR/VP16细胞中凋亡相关基因表达的改变可能是其产生多药耐药性的一个重要机制。

Objective： To establish an etoposide （VP16） resistant cell line JAR/VP16 of human choriocarcinoma, and to detect the biological characteristics of the cell, and to analyze the expression of its muhidrug resistance-and apoptosis-associated genes, so as to investigate a correlation between multidrug resistance and apoptosis-associated genes expression. Methods：The resistant cell line was obtained in vitro by culture of choriocarcinoma cell lineJAR with increasing stepwisely concentration gradient of VP16. Four months after the culture with the final concentration, the morphologic changes were observed with light-and electron-microscope, and the drug sensitivity to VP16, MTX, KSM, 5-FU, TAX, ADM and VCR were determined by MTT [3-（4,5-dimethyhhiazol-2-yl）-2,5-diphenyl tetrasodium bromide ] assay, respectively. Low flux cDNA microarray analyses about muhidrug resistance-and apoptosis-associated genes of JAR/VP16 and its parental cell line JAR were also performed. Results： ① JAR/VP16 cell line was established successfully after 14 months. Compared with the parental cell line, JAR/VP16 showed some great changes in morphology, with a high and a stable resistant index （RI） to VP16 of 36.78, JAR/VP16 cell line also exhibited a cross-resistance to some other chemotherapeutic agents, such as TAX （RI 26.18）, MTX （RI 18.67）, KSM （RI 14.73） besides VP16. ②The expression levels of some important multidrug resistance-associated genes such as MRP1, MRP2, MRP6, BCRP, LRP and Rb, were higher in JAR/VP16 than those in JAR. At the same time, the expression levels of some apoptosis-suppressing genes （Bcl-2, Bcl-x, MyD88, TRAF-4, TRAF-6, Trip, and so on） were raised and those of some apoptosis-promoting genes （Bax, BimL, Blk, Caspase-7,-8,-13,-14, Fas, FasL, and so on） decreased in JAR/VP16. Conclusion： JAR/VP16 acquired a muhidrug and stable resistance. The modulation of expression of apoptosis genes may be a main mechanism of acquired resistance in JAR/VP16.