P—gP在膀胱癌细胞中的差异表达研究

Differential expression of P-gp in bladder cancer cell lines

目的研究P-糖蛋白（P．gP）在人膀胱癌耐阿霉素细胞株（pumc-91／ADM）及不耐药膀胱癌细胞株（pumc-91）中的差异表达。方法横断面研究，采用阿霉素浓度梯度诱导法从人膀胱移行细胞癌puree-91建立人膀胱癌多药耐药细胞系pumc-91／ADM，通过SYBRGreenI实时荧光定量PCR（qRT-PCR）检测pumc-91／ADM和pumc-91细胞株中P—gPmRNA的表达情况，利用蛋白免疫印迹法（WesternBlot）对两种细胞中P-gP蛋白的表达进行半定量分析，同时采用免疫细胞化学技术对两种细胞中P-gP蛋白的表达进行定位及半定量分析，实验数据均使用独立样本的t检验进行分析。结果qRT-PCR结果显示，与pumc-91细胞相比，P-gPmRNA在pumc-91／ADM细胞中的表达水平上调了约7．74倍（t=11．97，P〈0．05）；利用Image J软件分析Westernblot条带光密度比值，P-gP蛋白在pumc-91／ADM中的表达量（1．393±0．328）明显高于pumc-91（0．953±0．250）（t=4．35，P〈0．05）；通过免疫细胞化学染色发现，P-gP在两种细胞的胞膜和胞浆均有表达，且在pumc-91／ADM中的表达量明显高于pumc-91（t=11．41，P〈0．05），差异均有统计学意义。结论P-gP在人膀胱癌耐阿霉素细胞株（pumc-91／ADM）中表达水平比其在不耐药膀胱癌细胞株（pumc-91）中表达水平显著上调。

Objective The generation of drug resistance often leads to the failure of the bladder cancer chemotherapy. P-glycoprotein （P-gp） is an ATP-dependent drug efflux pump linked to development of muhidrug resistance in cancer cells. The laboratory has successfully established adriamycin-resistant human bladder cancer cell line （pumc-91/ADM） from its parental cell line （pumc-91）. According to the drug resistant spectrum analysis, pumc-91/ADM cell line exhibited the characteristics of multi-drug resistance. However, the expression of P-gp in two cell lines was still unknown. In this paper, there was a comparison between pumc-91/ADM and puree-91 about the differential expression of P-gp. Methods To determine the expression and location of P-gp in pumc-91 and pumc-91/ADM, qRT-PCR, Western blot and immunocytochemistry were applied in the experiment, qRT-PCR was implemented to research the expression of P-gp mRNA in two cell lines （pumc-91/ADM and pumc-91 ）. Western blot was adopted to investigate the expression of P-gp protein in pumc-91 and pumc-91/ADM cell lines. Immunocytochemistry technique was used to explore the cellular location of P-gp and affirm its expression in two cell lines visually. Student＇s t-test was employed for statistical analysis and P 〈 0.05 was considered statistically significant. Results qRT-PCR analysis revealed that the expression of P-gp mRNA was upregulated in drug-resistant cell line pumc-91/ ADM compared to parental cell line pumc-91. To normalize for differences in the amount of total RNA, GAPDH was selected as an endogenous RNA control. Compared with pumc-91, the expression of P-gp mRNA was upregulated 7.74 fold in pumc-91/ADM （t = 11.97, P 〈 0. 05 ）. Consistent with the qRT-PCR result, Western blot confirmed the protein of P-gp expressed differentially in two cell lines. The expression of P-gp protein was significantly increased in pumc-91/ADM compared to pumc-91. According to the results, the differences between pumc-91 and pume-91/ADM had statistical significance （t = 4.35, P〈0.05）. Immunocytochemical analysis results demonstrated that P-gp was not only located in cell membrane but also in cytoplasm of the two cell lines. The expression of P-gp in pumc-91/ADM increased distinctly. The difference was statistically significant （ t = 11.41, P 〈 0. 05 ）. Conclusion Compared with pumc-91, the expression of P-gp in pumc-9l/ADM was significantly upregulated.