肝癌细胞HepG2间的P-糖蛋白传递作用

Intercellular transfer of P-glycoprotein in hepatocellular cell line HepG2

目的探讨肝癌细胞株HepG2细胞间是否存在P-糖蛋白（P—gp）的传递及P—gp与多药耐药基因（mdr1）的关系。方法将pSUPER．neo＋GFP质粒转染至HepG2细胞（命名为HepG2／GFP），并与阿霉素耐药HepG2细胞（命名为HepG2／ADM）混合培养。激光共聚焦显微镜观察肝癌细胞间P-gp的传递。免疫磁珠法分离混合培养细胞，收集原HepG2／GFP细胞（命名为HepG2／aqMDR），采用Western blot分析HepG2、HepG2／ADM、HepG2／GFP、HepG2／aqMDR细胞的P—gp表达水平，同时应用实时荧光定量PCR分析这些细胞mdr1 mRNA的表达水平。多样本均数比较用单因素方差分析，进一步两两比较用SNK—q检验。结果荧光显微镜下可见大量带绿色荧光的稳定克隆。激光共聚焦显微镜下见HepG2／aqMDR细胞以黄色荧光为主，而HepG2／GFP以绿色荧光为主，HepG2／ADM以红色荧光为主。Western blot检测结果显示HepG2／aqMDR细胞中P—gp表达水平低于HepG2／ADM，但明显高于HepG2／GFP（q=35．07，P〈0．05）与HepG2（q=36．87，P〈0．05）。实时荧光定量PCR结果显示，HepG2／ADM细胞中mdr1 mRNA表达水平较高，而HepG2／aqMDR、HepG2、HepG2／GFP三组细胞中mdr1的mRNA表达水平差异没有统计学意义（F=2．30，P〉0．05）。结论P—gp可以从耐药肝癌细胞传递至敏感肝癌细胞，这一现象为进一步研究肝癌多药耐药机制提供了新的思路。

Objective To investigate whether there is intercellular transfer of functional P-glycoprotein （P-gp） from P-gp-positive cells to P-gp-negative cells in vitro. Methods HepG2/GFP cells, a HepG2 cell line stably expressing GFP, were co-cultured with HepG2/ADM cells, an adriamycin-resistant cell line derived from HepG2 cells. The distribution of P-gp in hepatocellular carcinoma cell was observed under laser scanning confocal microscope （LSCM）. Immunomagnetic beads were used to separate HepG2/GFP cells from the mixed culture. The abundance of P-gp was analyzed by western blot, and the expression of mdr1 mRNA was detected by qRT-PCR. Results Yellow fluorescence was detected in HepG2/aqMDR cells, green fluorescence was detected in HepG2/GFP cells, red fluorescence was detected in HepG2/ADM cells by LSCM. The level of P-gp protein in HepG2/aqMDR cells was lower than that in HepG2/ADM cells, but higher than that in HepG2/GFP cells （q = 35.07, P 〈 0.05） and HepG2 cells （q = 36.87, P 〈 0.05）. The expression of mdrl mRNA in HepG2/ADM cells was higher than that in HepG2/aqMDR, HepG2 and HepG2/GFP cells, but there was no significant difference in mdrl mRNA among HepG2/aqMDR, HepG2 and HepG2/GFP cells （F = 2.30, P 〉 0.05）. Conclusions P-gp can transfer from drug resistant hepatocellular cells to sensitive hepatocellular carcimoma cells. This study suggests a novel mechanism of multidrug resistance in hepatocellular carcimoma.