CAJ | 학술논문

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pumc-91建立人膀胱癌多药耐药细胞系pumc-91/ADM,通过SYBR Green Ⅰ实时荧光定量PCR(qRT-PCR)检测pumc-91/ADM和pumc-91细胞株中P-gp mRNA的表达情况,利用蛋白免疫印迹法(Western Blot)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-gp mRNA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Western blot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)(=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.
목적 연구P-당단백(P-gp)재인방광암내아매소세포주(pumc-91/ADM)급불내약방광암세포주(pumc-91)중적차이표체.방법 횡단면연구,채용아매소농도제도유도법종인방광이행세포암pumc-91건립인방광암다약내약세포계pumc-91/ADM,통과SYBR Green Ⅰ실시형광정량PCR(qRT-PCR)검측pumc-91/ADM화pumc-91세포주중P-gp mRNA적표체정황,이용단백면역인적법(Western Blot)대량충세포중P-gp단백적표체진행반정량분석,동시채용면역세포화학기술대량충세포중P-gp단백적표체진행정위급반정량분석,실험수거균사용독립양본적t검험진행분석.결과qRT-PCR결과현시,여pumc-91세포상비,P-gp mRNA재pumc-91/ADM세포중적표체수평상조료약7.74배(t=11.97,P＜0.05);이용Image J연건분석Western blot조대광밀도비치,P-gp단백재pumc-91/ADM중적표체량(1.393±0.328)명현고우pumc-91(0.953±0.250)(=4.35,P＜0.05);통과면역세포화학염색발현,P-gp재량충세포적포막화포장균유표체,차재pumc-91/ADM중적표체량명현고우pumc-91(t=11.41,P＜0.05),차이균유통계학의의.결론 P-gp재인방광암내아매소세포주(pumc-91/ADM)중표체수평비기재불내약방광암세포주(pumc-91)중표체수평현저상조.
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 multidrug 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 pumc-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 pumc-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-91/ADM was significantly upregulated.