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背景与目的:部分非小细胞肺癌(non-smallcelllungcancer,NSCLC)表达野生型p53基因(wild-typep53,wt-p53),因此在基因治疗中克服这些NSCLC对wt-p53的抵抗机制就非常重要。P53基因家族的新成员p73是p53的同源体,本研究旨在探讨对wt-p53基因治疗抵抗的人肺腺癌细胞A549在外源p73基因转染或联用化疗药后凋亡程度和对化疗药物敏感性的变化。方法:将真核表达重组质粒pcDNA3-HA-p53或pcDNA3-HA-p73α转染入A549细胞,G418筛选,Westernblot检测P53或P73α的表达。MTT法分析转染细胞对顺铂和阿霉素的敏感性,用流式细胞术、TUNEL法和DNA片段法分析化疗药作用下转染细胞的凋亡变化,克隆形成实验观察细胞生物学性状的改变。结果:转染p53或p73α基因的A549细胞可以稳定高表达p53或p73α蛋白。转染p73α的A549细胞在原本没有明显抑制和杀伤作用的药物浓度(6.25μmol/L的顺铂或0.25μmol/L的阿霉素)作用下生长明显受到抑制,顺铂的IC50值从22.65μmol/L降至3.75μmol/L,阿霉素的IC50值从4.20μmol/L降至0.06μmol/L。p73能使A549细胞受顺铂和阿霉素诱导的细胞凋亡增加,而p53没有明显作用。流式细胞术显示p73α基因转染后,顺铂诱导的细胞凋亡率从10.6%升高到36.8%(P<0.01),阿霉素诱导的细胞凋亡率从13.0%升高到41.1%(P<0.01)。克隆形成实验显示,p73α基因转染能明显降低顺铂和阿霉素作用后A549细胞的克隆形成数(P<0.01),对顺铂和阿霉素的化疗增效倍数分别为2.0和2.4倍。结论:外源性p73基因的导入增加了wt-p53型A549细胞对顺铂和阿霉素等化疗药的敏感性,该作用可能与p73基因能不依赖于p53基因诱导细胞凋亡有关。p73基因可用于治疗wt-p53不能发挥作用的恶性肿瘤。
BACKGROUND & AIM: Non-small cell lung cancer (NSCLC) expresses wild-type p53 (wt-p53), so it is important to overcome the resistance mechanism of wt-p53 in gene therapy. P53 gene family of a new member of p73 is a p53 homologue of the purpose of this study is to explore the wt-p53 gene therapy resistant human lung adenocarcinoma cell line A549 in vitro p73 gene transfection or combined with chemotherapy after the degree of apoptosis and Changes in chemosensitivity to chemotherapy. Methods: The eukaryotic expression plasmid pcDNA3-HA-p53 or pcDNA3-HA-p73α was transfected into A549 cells. The cells were screened by G418 and the expression of P53 or P73α was detected by Western blot. The sensitivity of transfected cells to cisplatin and doxorubicin was analyzed by MTT assay. The apoptosis of transfected cells was analyzed by flow cytometry, TUNEL and DNA fragment analysis. Clonal formation assay was used to observe the cell biological characteristics Change. Results: A549 cells transfected with p53 or p73α gene could stably overexpress p53 or p73α protein. A549 cells transfected with p73α were significantly inhibited by the drug concentration (6.25μmol / L cisplatin or 0.25μmol / L doxorubicin) without obvious inhibition and killing effect. The IC50 value of cisplatin was decreased from 22.65μmol / L decreased to 3.75μmol / L, doxorubicin IC50 value decreased from 4.20μmol / L to 0.06μmol / L. p73 can induce apoptosis of A549 cells induced by cisplatin and doxorubicin, while p53 has no obvious effect. Flow cytometry showed that cisplatin-induced apoptosis increased from 10.6% to 36.8% (P <0.01) after transfected with p73α gene, and the apoptosis rate induced by doxorubicin increased from 13.0% to 41.1% (P <0.01). Clone formation assay showed that the transfection of p73α gene could significantly reduce the number of clone formation of A549 cells after cisplatin and doxorubicin treatment (P <0.01), and the synergistic multiplication rates of cisplatin and doxorubicin were 2.0 and 2.4 times . Conclusion: The introduction of exogenous p73 gene increases the sensitivity of wt-p53 A549 cells to chemotherapy drugs such as cisplatin and doxorubicin, which may be related to the fact that p73 gene can not induce cell apoptosis induced by p53 gene. The p73 gene can be used to treat malignant tumors where wt-p53 does not work.