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目的 探讨p53基因突变与肺癌化疗后获得性耐药的关系。方法 采用顺铂(DDP)大剂量(50μmol/L)间歇诱导法,体外诱导具有野生型p53基因的人大细胞肺癌NCIH460细胞株,建立多药耐药细胞系H460/DDP;用免疫细胞化学法检测P糖蛋白(Pgp)、多药耐药相关蛋白(MRP1)、肺相关蛋白(LRP)、拓扑异构酶Ⅱ(TOPOⅡ)、谷胱甘肽转移酶π(GSTπ)及P53蛋白的表达状况;用荧光免疫法测定P53蛋白磷酸化状态;对p53cDNA全长进行扩增及测序,用含野生型p53基因的质粒pShuttleCMVwtp53cDNA转染耐药细胞,测定转染细胞的药物敏感性。结果 新建立的人大细胞肺癌多药耐药株H460/DDP,对DDP及卡铂的耐药指数分别为1021和998,对氟尿嘧啶、多柔比星、表柔比星、甲氨蝶呤、依托泊苷及异长春新碱等有不同程度的交叉耐药。多药耐药株H460/DDP细胞的P53蛋白滞留于细胞浆,在DDP刺激下不能发生磷酸化;LRP表达明显增加(P<005);但其他耐药相关蛋白水平与DDP诱导前相比差异无统计学意义(P>005);耐药株细胞p53基因第277位点后插入一个“t”;耐药株转染pShuttleCMVwtp53cDNA后,其耐药性可发生部分(532%)逆转。结论 铂类化疗药物诱导的NCIH460细胞p53基因突变与其对化疗药物耐药性的产生有密切关系,p53基因替代疗法可能是克服这类获得性耐药的一个有效方
Objective To investigate the relationship between p53 gene mutation and acquired drug resistance after chemotherapy in lung cancer. Methods Human large cell lung cancer NCIH460 cell line with wild-type p53 gene was induced in vitro by intermittent induction with DDP (50μmol / L) to establish multidrug-resistant cell line H460 / DDP. Immunocytochemistry Pgp, MRP1, LRP, TOPOⅡ, GSTπ and P53 protein expression were detected by Western blot. The phosphorylation of P53 protein was detected by fluorescence immunoassay. The full length of p53 cDNA was amplified and sequenced. The drug-resistant cells were transfected with plasmid pShuttleCMVwtp53cDNA containing wild-type p53 gene. The drug sensitivity of transfected cells was determined. Results The newly established human large cell lung cancer multi-drug resistant strain H460 / DDP, resistance index of DDP and carboplatin were 1021 and 998, respectively, for fluorouracil, doxorubicin, epirubicin and methotrexate Polymorphic acid and vincristine have different degrees of cross-resistance. P53 protein in H460 / DDP cells resided in the cytoplasm and could not be phosphorylated under the stimulation of DDP; LRP expression was significantly increased (P <005); however, the levels of other drug-resistance-related proteins were different from those before DDP induction There was no statistical significance (P> 005). A “t” was inserted after the 277th site of the p53 gene in the drug-resistant cell line. The resistance of the drug-resistant cell transfected with pShuttleCMVwtp53cDNA was partially reversed (532%). Conclusion The mutation of p53 gene in NCIH460 cells induced by platinum chemotherapeutic drugs is closely related to its resistance to chemotherapeutic drugs. P53 gene replacement therapy may be an effective way to overcome such acquired drug resistance