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

The establishment and biological characteristics of noscapine resistant human colon carcinoma cell line HT-29/Nos,LoVo/Nos and SW480/Nos

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、LoVo/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(IC_(50))。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G_1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. ICs0 of noseapine 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. Results 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）hvs. （38.38＋1.88）h, P〈0.05; and （48.84 ±1.40）h vs. （31.30± 1.75 ） h, P 〈 0.05 ） ]. Flow eytometry demonstrated that the phase GI was 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.