目的 探讨内质网应激激活与拓扑异构酶Ⅱα介导的依托泊甙( etoposide,VP16)和放线菌素D(actinomycin-D,Act-D)耐药绒癌的关系.方法 采用间断诱导法诱导人绒癌细胞系JEG-3,分别建立绒癌ActD耐药细胞系JEG-3/ActDB1和VP16耐药细胞系JEG-3/VPC1.细胞计数法(cell counting kit-8,CCK-8)观察敏感细胞和耐药细胞的生长增殖规律和耐药指数；流式细胞仪检测细胞周期比例和染色体倍性变化；荧光定量PCR技术分别检测两种不同药物诱导的JEG-3耐药株中拓扑异构酶Ⅱα(TopoⅡα)和内质网应激(ERS)各个通路相关的基因mRNA的表达水平.结果 (1)成功建立了绒癌耐药细胞系JEG-3/ActDB1和JEG-3/VPC1.其耐药指数分别为50.64±10.68和65.87±3.19.耐药株生长速度均较亲本细胞减慢,三者的染色体倍性差异无统计学意义.(2)在两种不同的耐药株中,TopoⅡα的表达水平均明显低于亲本细胞,而与ERS各个通路相关基因均有不同程度的激活.结论 成功建立了针对ActD和VP16耐药的绒癌细胞系JEG-3/ActDB1和JEG-3/VPC1.内质网应激激活可能参与绒癌耐药的发生.%Objective To investigate the relationship of endoplasmic reticulum stress (ERS) and topoisomerase Hot (TopoIIa) expression in the mechanism of actinomycin-D (ActD) and etoposide (VP16)-resistant human choriocarcino-ma. Methods The ActD-and VP-resistant sub-line were established by intermitted exposure to grads increased chemotherapy reagent respectively. Population doubling time was calculated and compared based on the growth curve of these cell lines, cell cycles and chromosomal ploidy were assayed with flowcytometry methods. The resistant index (RI) was measured by Cell Counting Kit-8 ( CCK-8) assay. qRT-PCR was used to detect the mRNA expression level of TopoIIa and the genes involved in the pathway of ERS. Results (1) The RI of JEG-3/ActDB, and JEG-3/VPC; were 50. 64 ± 10. 68 and 65. 87 ±3. 19 respectively. Compared with the JEG-3 cell line, the chemo-resistant sub-line had gross changes in morphological, cell growth and cell cycles. (2) The transcript expression level of TopoIIa were significant down-regulated in chemo-resistant sub-lines, while the genes involved in ER-related pathway were changed to certain extension by comparing with parent cell line and chemo-resistance sub-lines. Conclusion We successfully established the ActD-and VP16-resistant sub-line of JEG-3 respectively. The activation of ERS related pathway may involved in chemo-resistance of choriocarcinoma.
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