【摘要】 目的探讨鼻咽癌PTEN和survivin基因与西妥昔单抗耐药的相关性。方法以人鼻咽癌细胞株5-8F细胞为研究对象,采用逐步增加剂量法诱导建立西妥昔单抗耐药细胞5-8F/Erbitux;MTT法检测西妥昔单抗对细胞的半数抑制浓度IC50,计算耐药指数(RI);计数法描绘生长曲线,计算倍增时间;流式细胞仪检测细胞周期;Western blot法检测细胞PTEN、survivin的表达;实时荧光定量PCR(QPCR)检测细胞PTEN、survivin基因的表达量;测序方法检测细胞PTEN基因第5、7、8外显子是否突变。结果建立西妥昔单抗耐药的人鼻咽癌细胞系5-8F/Erbitux,西妥昔单抗作用3天和5天的RI分别为1.2和1.1,5-8F与5-8F/Erbitux细胞的倍增时间分别为26.63h和142.30h;5-8F/Erbitux的G0/G1期比例显著增高(P<0.001),S期比例显著下降(P<0.001),G2/M期比例下降,但差异不显著(P>0.05);5-8F/Erbitux细胞PTEN蛋白表达水平显著升高(P<0.001),survivin蛋白表达水平显著下降(P<0.001);survivin基因表达量显著下降(P=0.000),PTEN基因表达量有所升高,但差异不显著(P=0.085)。通过比对,5-8F/Erbitux细胞PTEN基因第5、7、8外显子未见突变。结论西妥昔单抗可通过增强PTEN的表达和降低survivin的表达抑制鼻咽癌细胞增殖。鼻咽癌PTEN缺失或突变以及survivin过表达与西妥昔单抗耐药无关。更多还原

【Abstract】 Objective To explore the correlation of PTEN and survivin gene and resistance of nasopharyngeal carcinoma to cetuximab.Methods The nasopharyngeal cancer cell line 5-8F which was epidermal growth factor receptor(EGFR) high expressive was screened.Cetuximab-resistant 5-8F/Erbitux was induced by stepwise selection after exposure to increasing doses of cetuximab.IC50 was determined by MTT assay and the resistance index(RI) was calculated.Cell growth curves were plotted and the double times were calculated by cell counting assay.Distribution of cell cycles were detected by flow cytometry.Expression levels of PTEN and survivin protein were determined by Western blot method and PTEN and survivin gene by real-time fluorescent quantitation PCR.The 5,7 and 8 exons of PTEN gene were detected by sequencing method.Results A cetuximab-resistant human nasopharyngeal carcinoma cell line 5-8F/Erbitux established successfully and the RI were 1.2 and 1.1 respectively with the action time of cetuximab were 3 and 5 days.The doubling times of 5-8F and 5-8F/Erbitux cells were 26.63 and 142.30h,respectively.Flow cytometry demonstrated that 5-8F/Erbitux cells of phase G0/G1 increased significantly(P<0.001),and those of S and G2/M phase reduced,but the difference of the former was significant(P<0.001) and the latter was not significant(P>0.05).5-8F/Erbitux had a higher expression of PTEN protein(P<0.001) but the survivin decreased significantly(P<0.001).The quantity of survivin gene decreased significantly(P=0.000)and PTEN gene increased,but not significant(P=0.085).There was not any mutation of 5,7 and 8 exons of PTEN gene in 5-8F/Erbitux through contradistinction.Conclusion Cetuximab could inhibit the proliferation of nasopharyngeal cancer cell through upgrade the expression of PTEN and degrade of survivin.There is not any relationship between the absence or mutation of PTEN and the resistance of nasopharyngeal carcinoma to cetuximab,as well as the overexpression of survivin.更多还原