对培美曲塞耐药胰腺癌Patu8988细胞株克隆、侵袭能力及相关耐药基因的研究_临床医学论文.doc

对培美曲塞耐药胰腺癌Patu8988细胞株克隆、侵袭能力及相关耐药基因的研究_临床医学论文 对培美曲塞耐药胰腺癌Patu8988细胞株克隆、侵袭能力及相关耐药基因的研究_临床医学论文 作者：杨军，石欣,严伟,林士波,顾海涛,钱程佳 【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500 μmol·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500 μmol·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。 【关键词】 胰腺肿瘤； 获得性耐药； 培美曲塞； ABC多药转运蛋白G家族成员2 　　［Abstract］ Objective:To investigate the cloning capability,invasion and associated multian 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).RT20%.Immunofluorescence showed drugresistant cells of ABCG2 stronger fluorescence intensity than their parental cells.Laser confocal detection of drugresistant cells showed ABC