摘要
目的采用微流控芯片技术分析细胞外酸性环境对肿瘤细胞P-糖蛋白(P-gp)表达、活性及其介导的道诺霉素细胞毒性的影响。方法在微流控芯片上将A549细胞分为实验组和对照组,实验组用p H为6.6酸性细胞培养液处理,对照组用p H为7.4中性培养液常规培养,芯片上进行细胞免疫荧光技术分析P-gp表达,罗丹明123外排实验评价P-gp活性,细胞存活/凋亡荧光染色法分析道诺霉素的细胞毒性。结果微流控芯片可为A549细胞生长提供适宜的微环境,细胞在72 h后融合度能达到90%以上。酸性细胞培养液处理对P-gp表达未产生显著影响,但可显著增强P-gp活性,处理时间6 h时A549细胞P-gp活性达到峰值。酸性细胞培养液处理6 h后道诺霉素细胞毒性效率显著降低,在P-gp抑制剂维拉帕米协同作用下道诺霉素细胞毒性得到逆转。结论微流控芯片技术可缩短分析时间,降低试剂耗量,为进一步认识肿瘤多药耐药机制和高效肿瘤耐药逆转剂筛选提供新的技术平台。
Objective To explore the impact of extracellular acidic environment on the expression and activity of P-glycoprotein（ P-gp） and on the P-gp-mediated cytotoxicity of daunomycin in cancer cells by using microfluidic chip technology. Methods The A549 cells cultured on a microfluidic chip were divided into experiment group and control group. The experiment group was exposed to an acidic cell culture medium（ p H 6. 6）,while the control group was treated with a neutral cell culture medium（ p H 7. 4）. The expression of P-gp was detected by cell immunofluorescense analysis and the activity of P-gp was evaluated by Rhodamine 123 efflux experiment. Meanwhile,the cytotoxicity of daunomycin was analyzed by cell live / dead fluorescence staining meth-od. Results Microfluidic chip designed in this study could provide a suitable microenvironment for the growth of A549 cells and the A549 cells reached the confluence of 90% after inoculation for 72 h. Treatment of the acidic cell culture media on A549 cells did not make a significant difference on the expression level of P-gp. However,the activity of P-gp was significantly enhancement and peaked at 6 h after treatment with acidic cell culture media. Meanwhile,the cytotoxicity of daunomycin reduced significantly after treatment with acidic cell culture medium for 6 h,and a reversal effect was obtained when synergy with verapamil. Conclusions Microfluidic chip technology can shorten the analysis time and reduce the reagent consumption. It can be used as a new technology platform for understanding the mechanisms of multi-drug resistance and for screening highly efficient multidrug resistance reversal agents.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2015年第1期75-81,共7页
Acta Academiae Medicinae Sinicae
基金
重庆市自然科学基金面上项目(cstc2012jj A10046)
重庆市卫生局医学科研计划项目(2012-2-165)
重庆市永川区科技计划项目(YCSTC
2012BE5002)~~
关键词
多药耐药
酸性细胞外环境
P-糖蛋白
微流控芯片
细胞毒性
multi-drug resistance
acidic extracellular environment
P-glycoprotein
microfluidic chip
cytotoxicity
