人结肠癌那可丁耐药细胞株HT-29/Nos、LoVo/Nos、SW480/Nos的构建及其生物特性探究

摘要

目的 构建那可丁(Nos)耐药的人结肠癌细胞株(HT-29/Nos、LoVo/Nos、SW480/Nos),探究细胞株的生物学特性.方法 采用反复大剂量结合药物浓度梯度法建立人结肠癌耐药细胞株HT-29/Nos、Lo-Vo/Nos、SW480/Nos.CCK-8法检测Nos对人结肠癌细胞株 HT -29、LoVo、SW480和耐药细胞株 HT-29/Nos、LoVo/Nos、SW480/Nos细胞的半数抑制率(IC50).细胞生长曲线的测定.流式细胞术测定各组细胞株的细胞周期变化.Real time PCR和Western blot分别检测耐药相关基因和蛋白的表达.结果 成功构建人结肠癌耐药细胞株HT-29/Nos、LoVo/Nos、SW480/Nos,耐药指数分别为6.37、7.15、6.81.HT-29/Nos细胞倍增时间较HT-29显著增长[(51.42 ±2.83)h vs(35.05 ±3.61)h,P<0.05];LoVo/Nos细胞倍增时间较LoVo显著增长[(61.93 ±4.22)h vs(38.38 ±1.88)h,P<0.05];SW480/Nos细胞倍增时间较SW480增长[(48.84 ±1.40)h vs(31.30 ±1.75)h,P<0.05].耐药细胞株HT-29/Nos、LoVo/Nos、SW480/Nos的G1期细胞数目显著增加(P<0.05),S期细胞数目显著减少(P<0.05).耐药细胞株HT-29/Nos、LoVo/Nos、SW480/Nos的P-gp和MDR-1 mRNA和蛋白表达均高于HT-29、LoVo、SW480细胞株.结论 成功构建了Nos耐药人结肠癌细胞株HT-29/Nos、LoVo/Nos、SW480/Nos,为下一步耐药性分子机制的研究奠定基础.%Objective To establish the noscapine(Nos)resistanct human colon cancer cell lines and study their biological characteristics.Methods HT-29/Nos, LoVo/Nos and SW480/Nos cell lines were established by stepwise selection in increasing dose of noscapine.IC50of noscapine in HT -29, LoVo, SW480, HT-29/Nos, LoVo/Nos and SW480/Nos cell lines were detected by CCK -8.Cell growth curve was established and the doubling time was accounted. The cell cycle distribution was determined by flow cytometry.The mRNA and protein expression levels of P -gp and MDR-1 in human colon cancer cell and resistant colon cancer cell lines were examined by RT -PCR and Western blot.Re-sults The noscapine resistant human colon cancer cell line HT -29/Nos,LoVo/Nos and SW480/Nos were successfully established,with the resistance indexes of 6.37,7.15 and 6.81,respectively.The doubling times of HT -29/Nos,Lo-Vo/Nos and SW480/Nos were obviously longer than those of HT -29, LoVo and SW480 cell[(51.42 ±2.83)h vs. (35.05 ±3.61)h,P<0.05;(61.93 ±4.22)h vs.(38.38 ±1.88)h,P<0.05; and(48.84 ±1.40)h vs.(31.30 ± 1.75)h,P<0.05)].Flow cytometry demonstrated that the phase G 1was increased and phase S was shortened in drug resistant colon cancer cells.Both mRNA and protein expression levels of P -gp and MDR-1 in HT-29/Nos,LoVo/Nos and SW480/Nos cell lines were higher than those in HT -29,LoVo and SW480.Conclusion We have successfully es-tablished cell line HT -29/Nos,LoVo/Nos and SW480/Nos with Nos resistance,which lays a foundation for the study of resistance mechanism.