[甲氨蝶呤耐药人绒毛膜癌细胞系的建立及其生物学特性]

[Establishment of methotrexate-resistant human choriocarcinoma cell line and its biological characteristics].

作者信息

Chen Ya-xia, Xia Xing, Chen Huai-zeng, Cheng Qi, Zhou Cai-yun

机构信息

The Affiliated Obstetrics and Gynecology Hospital, College of Medicine Zhejiang University, Hangzhou 310006, China.

出版信息

Zhejiang Da Xue Xue Bao Yi Xue Ban. 2004 Mar;33(2):138-42, 165. doi: 10.3785/j.issn.1008-9292.2004.02.011.

DOI:10.3785/j.issn.1008-9292.2004.02.011

PMID:15067735

Abstract

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.

摘要

目的

建立甲氨蝶呤（MTX）耐药的绒毛膜癌细胞系并确定其生物学特性。

方法

通过将人绒毛膜癌细胞系JAR暴露于间歇且逐渐增加浓度的MTX中，获得MTX耐药细胞系（JAR/MTX）。采用MTT法检测药物敏感性；免疫组织化学法检测P-糖蛋白（P-gp）、谷胱甘肽S-转移酶-π（GST-Pi）和增殖细胞核抗原（PCNA）的表达。采用碘化丙啶（PI）/膜联蛋白V（Annexin V）染色，通过流式细胞术（FCM）检测细胞凋亡。同时测量生长速率和人绒毛膜促性腺激素（HCG）的分泌量。

结果

成功建立了JAR/MTX细胞系，其对MTX具有稳定耐药性（MTX耐药指数为７.３），并对紫杉醇（TAX）和长春新碱（VCR）具有交叉耐药性。JAR/MTX的生长速率低于亲代细胞系JAR。JAR/MTX中PCNA的表达水平低于JAR（分别为3.09±0.42和3.72±0.35，P<0.05），而GST-Pi的表达较高。两细胞系间P-gp表达无统计学差异。48小时后，每10(5)个JAR/MTX细胞分泌的HCG量比JAR多（分别为95.7±5.４和41.3±2.8）mIU（P<0.01）。流式细胞术显示，JAR/MTX的自发凋亡及MTX诱导的凋亡均显著低于JAR（P<0.05）。