人膀胱癌顺铂耐药细胞株T24／CDDP的建立和鉴定及耐药机制研究

Establishment of cisplatin-resistant human bladder cancer cell line and investigation of its resistant mechanism

目的 建立人膀胱癌顺铂（CDDP）耐药细胞株T24/CDDP,鉴定其生物学特性,并探讨其对CDDP耐药的机制.方法 采用CDDP浓度逐步递增联合大剂量CDDP冲击方法,在体外连续诱导、培养膀胱癌细胞株124细胞,建立对CDDP耐药的细胞株T24/CDDP.CCK-8法测定T24/CD-DP细胞对CDDP的耐药指数;绘制细胞生长曲线,计算细胞群体倍增时间;流式细胞术检测T24细胞和124/CDDP细胞的细胞周期比例;Western blot测定细胞中蛋白激酶Cα（PKCα）和P-糖蛋白（P-gp）蛋白的表达.结果 124细胞在体外采用CDDP浓度逐步递增联合大剂量CDDP冲击方法连续诱导、培养,历时11个月,成功建立了对CDDP耐药的细胞株T24/CDDP,其耐药指数为4.17.T24细胞和T24/CDDP细胞的群体倍增时间分别为（33.61±0.41）h和（39.94 ±0.63）h,两者比较差异有统计学意义（t=14.59,P=0.00）.与124细胞相比,T24/CDDP细胞的S期细胞比例减少[（30.63±2.74）％ vs （35.38±3.31）％],但差异无统计学意义（P＞0.05）.G0/G1期细胞比例增加[（60.42±3.25）％ vs （47.25±4.17）％],G2/M期细胞比例减少[（8.95±2.81）％ vs （17.37±3.46）％],差异均有统计学意义（t=4.31,P=0.006;t=3.27,P=0.015）.T24细胞和124/CD-DP细胞中均有PKCα和P-gp蛋白的表达.与T24细胞相比较,124/CDDP细胞中PKCα和P-gp蛋白表达水平均显著升高[（3.12±0.11）vs （1.37±0.06）,t=24.19,P=0.00;（1.98±0.08） vs（0.47±0.03）,t=30.61,P=0.00].结论 T24/CDDP细胞具有耐药表型及耐药细胞的生物学特性,其耐药机制可能与细胞中PKCα和P-gp蛋白的过度表达有关.

Objective To establish the cisplatin （CDDP）-resistant cell line from human bladder cancer cell line T24 and to investigate its resistant mechanism to CDDP.Methods A CDDP-resistant bladder cancer cell line T24/CDDP was established by gradually increasing dose of cisplatin and high-dose stimulation.The IC50 and resistance index were estimated with cell counting Kit-8 （CCK-8）.Cell growth curve, doubling time, and cell cycle phase distribution were measured.The expressions of protein kinase Cα （PKCα） and P-glycoprotein （P-gp） of T24 cells and T24/CDDP cells were measured with Western blot.Results The CDDP-resistant cell line T24/CDDP was established after 1 1 months, with a stable resistance to cisplatin and a resistant index of 4.17.The doubling time of T24 cells and T24/CDDP cells was（33.61 ±0.41）h and （39.94±0.63） h, with a significant difference （t =14.59, P =0.00）.Compared to the S phase of T24 cells [（30.63 ± 2.74） %], the S-phase of T24/CDDP cells was increased [（35.38 ±3.31）%], without significant difference （P 〉0.05）.The G0/G1 phase of T24/CDDP cells [（60.42 ±3.25）%] was significantly more than that of T24 cells [（47.25 ±4.17）%], and the G2/M phase of rT24/CDDP cells [（8.95 ±2.81）%] was significantly less than that of T24 cells [（17.37 ±3.46）%] （t =4.31, P =0.006;t =3.27, P =0.015）.Western blot showed that expressions of PKCα and P-gp in T24/CDDP cells were significantly higher than T24 cells [（3.12 ± 0.11） vs （1.37 ± 0.06）, t =24.19, P =0.00;（1.98 ±0.08） vs （0.47 ±0.03）, t =30.61, P =0.00].Conclusions T24/CDDP cell line showed a typical resistant phenotype and biological characteristics, which may be related to the overexpression of PKGα and P-gp protein.