人绒毛膜癌甲氨蝶呤耐药细胞株的建立及其特性的研究

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(3.09±0.42 vs 3.72 ± 0.35, P<0.05).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48 h后每105个细胞分泌HCG分别为(95.7±5.4 vs 41.3±2.8)mIU(P＜0.01).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<0.05).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.

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

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 sensitivity was detected by MTT; P-gp GST-Pi 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 compared with 3.72+/-0.35, P<0.05), while GST-pi expression was higher. No statistical difference of P-gp expression existed between two cell lines. JAR/MTX secreted more HCG than JAR every 10(5) cells secreted (95.7+/-5.4 compared with 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.