人绒毛膜癌甲氨蝶呤耐药细胞株的建立及其特性的研究

Establishment of methotrexate-resistant human choriocarcinoma cell line and its biological cha-racteristics

目的 :建立耐甲氨蝶呤 (MTX)的人绒毛膜癌细胞株 ,并检测其生物学特性。方法 :采用剂量递增间歇诱导的方法 ,对人绒毛膜癌细胞株 JAR进行长期诱导培养 ,建立耐药细胞株 JAR/ MTX。比较两种细胞倍增时间、绒毛膜促性腺激素 (HCG)分泌量、对 MTX和一些常用化疗药的敏感性 ,P-糖蛋白、谷胱甘肽 S转移酶、核增殖抗原的表达 ,以及细胞自发和 MTX诱导调亡率。结果 :历时 1年建成 JAR/ MTX细胞株 ,耐药性稳定 ,耐药指数 7.3,对紫杉醇、长春新碱有交叉耐药。 JAR/ MTX细胞增殖缓慢 ,核增殖抗原表达低于 JAR细胞 (3.0 9± 0 .4 2 vs 3.72±0 .35 ,P<0 .0 5 )。谷胱甘肽 S转移酶表达高于 JAR,P-糖蛋白的表达两者无差异。 HCG分泌高于 JAR细胞 ,培养4 8h后每 10 5个细胞分泌 HCG分别为 (95 .7± 5 .4 vs 4 1.3± 2 .8) m IU(P<0 .0 1)。 JAR/ MTX细胞的自发凋亡及MTX诱导的凋亡均明显少于其母系 JAR细胞 (P<0 .0 5 )。结论 :JAR/ MTX细胞对 MTX耐药性稳定 ,具有独特的细胞形态、染色体核型、增殖与凋亡等特征 ,为进一步研究绒癌的耐药机制提供了理想的细胞模型。

Objective: To establish a methotrexate (MTX)-resistant choriocarcinoma cell line and to determine its biologic characteristics. Methods: MTX-resistant cell line (JAR/MTX) was derived from human choriocarcinoma cell line JAR by exposed to intermittently and progressively increasing concentration of MTX. Drug sensiti-vity was detected by MTT; P-gp,GST-π and PCNA expressions were detected by immunohistochemistry. Cell apoptosis was detected by flow cytometry (FCM) with PI/Annexin V stain. Growth rates and human chorionic gonadotropin (HCG) production were also measured. Results: JAR/MTX cell line was established with stable MTX-resistance (resistance index to MTX was 7.3) and cross-resistant to TAX and VCR. Growthrate of JAR/MTX was lower than that of parent cell line JAR. Expression level of PCNA in JAR/MTX was lower than that in JAR (3.09±0.42 vs 3.72±0.35, P<0.05), while GST-π expression was higher. No statistical difference of P-gp expression existed between two cell lines. JAR/MTX secreted more HCG than JAR, every 105 cells secreted (95.7±5.4 vs 41.3±2.8)mIU after 48 h(P<0.01). The flow cytometry showed that the spontaneous and MTX-induced apoptosis in JAR/MTX was significantly lower than that in JAR P<0.05. Conclusion: JAR/MTX cell line presented stable resistant to MTX and cross-resistant to TAX and VCR, which might sever as a model in study of drug resistance in choriocarcinoma.