中文摘要：

目的：探讨对培美曲塞耐药的胰腺癌Patu8988细胞株相对于亲本株在细胞克隆、侵袭能力及多药耐药相关蛋白1（MDR1）、ABC多药转运蛋白G家族成员2（ABCG2）、ABC多药转运蛋白C家族成员3（ABCC3）耐药基因的变化。方法：采用双层软琼脂克隆实验分别在无药及加入500μmol·L^-1培美曲塞的环境下测定胰腺癌Patu8988耐药株及亲本株克隆生成能力的改变;采用Transwell侵袭小室检测两种条件下胰腺癌Patu8988侵袭能力的差异;采用RT-PCR法检测胰腺癌Patu8988细胞MDR1、ABCG2、ABCC3的mRNA表达水平,Wersten blot检测ABCG2和ABCC3的蛋白表达水平,免疫荧光及激光共聚焦检测ABCG2的表达及分布。结果：在无药及加入500μmol·L^-1培美曲塞的环境下,对培美曲塞耐药胰腺癌Patu8988细胞株的增殖克隆能力均与其亲本株有差异（P〈0.05）,而两种培养条件下胰腺癌Patu8988细胞株的侵袭能力变化差异无统计学意义（P〉0.05）。RT-PCR检测显示,在耐药株中ABCG2、ABCC3 mRNA表达分别是其亲本株的2.5倍和1.6倍左右,而MDR1 mRNA表达仅提高到1.2倍;Werstenblot结果显示蛋白表达为其亲本株的2.4倍和1.5倍。免疫荧光检测结果显示,耐药株中ABCG2的荧光强度较强,而其亲本株细胞较弱。激光共聚焦显示,耐药株中ABCG2表达主要分布于细胞膜,细胞质内也有少量分布。结论：相对于亲本株,对培美曲塞耐药的胰腺癌Patu8988细胞株克隆能力加强,而侵袭能力无明显改变。多药耐药基因表达均有不同程度的提高,以ABCG2基因表达提高尤为明显,而且其在耐药株中主要分布在细胞膜,细胞质内也有分布。

英文摘要：

Objective：To investigate the cloning capability,invasion and associated multi-drug resistance genes：MDR1（P-gp）,ABCG2,ABCC3 of Patu8988 human pancreatic cancer cells resistance to the pemetrexed compared with it′s parental strain.Methods：The cloning capability of Patu8988 human pancreatic cancer and it′s parental strain were detected in condition free or adding 500 μmol·L^-1 pemetrexed by soft agar cloning method.The invasion ability betweent them were detected by transwell invasion chamber.The mRNA expression of MDR1,ABCG2 and ABCC3 were determined by RT-PCR,ABCG2 and ABCC3 protein expression levels were detected by Wersten blot,the expression and distribution of ABCG2 were detected by immunofluorescence and laser scanning confocal microscopy.Results：The cloning capability between drug-free and adding 500 μmol·L^-1 pemetrexed was significantly different（P〈0.05）,whereas there was no significant changes of the invasion ability between them（P〉0.05）.RT-PCR and Wersten blot showed the mRNA and protein levels of ABCG2,ABCC3 were almost 250% and 150% in the drug-resistant cells than their parental cells,while the expression of MDR1 mRNA increased only 20%.Immunofluorescence showed drug-resistant cells of ABCG2 stronger fluorescence intensity than their parental cells.Laser confocal detection of drug-resistant cells showed ABCG2 expression was mainly distributed in the cell membrane,also a small amount of the distribution.Conclusion：The cloning capability is significantly different between Patu8988 human paucreatic cancer cells resistance to the pemetrexed and it′s parental strain,whereas invasion ability is no changes.ABCG2 is particularly increase than other multi-drug resistance genes and it is mainly distributed in the cell membrane and distribution.