中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2009年
11期
839-842
,共4页
陈龙%左国庆%吴进峰%曾贵利%李涛%刘作金%唐开福
陳龍%左國慶%吳進峰%曾貴利%李濤%劉作金%唐開福
진룡%좌국경%오진봉%증귀리%리도%류작금%당개복
癌%肝细胞%P糖蛋白%基因%MDR%抗药性%多药
癌%肝細胞%P糖蛋白%基因%MDR%抗藥性%多藥
암%간세포%P당단백%기인%MDR%항약성%다약
Carcinoma,hepatocellular%P-glycoprotein%Genes,mdr%Drug resistance,multiple
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(mdrl)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mdrl 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mdrl mRNA表达水平较高,而HepG2/aqMDR、HepG2、HepG2/GFP三组细胞中mdrl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.
目的 探討肝癌細胞株HepG2細胞間是否存在P-糖蛋白(P-gp)的傳遞及P-gp與多藥耐藥基因(mdrl)的關繫.方法 將pSUPER.neo+GFP質粒轉染至HepG2細胞(命名為HepG2/GFP),併與阿黴素耐藥HepG2細胞(命名為HepG2/ADM)混閤培養.激光共聚焦顯微鏡觀察肝癌細胞間P-gp的傳遞.免疫磁珠法分離混閤培養細胞,收集原HepG2/GFP細胞(命名為HepG2/aqMDR),採用Western blot分析HepG2、HepG2/ADM、HepG2/GFP,HepG2/aqMDR細胞的P-gP錶達水平,同時應用實時熒光定量PCR分析這些細胞mdrl mRNA的錶達水平.多樣本均數比較用單因素方差分析,進一步兩兩比較用SNK-q檢驗. 結果熒光顯微鏡下可見大量帶綠色熒光的穩定剋隆.激光共聚焦顯微鏡下見HepG2/aqMDR細胞以黃色熒光為主,而HepG2/GFP以綠色熒光為主,HepG2/ADM以紅色熒光為主.Western blot檢測結果顯示HepG2/aqMDR細胞中P-gp錶達水平低于HepG2/ADM,但明顯高于HepG2/GFP(q＝35.07,P<0.05)與HepG2(q=36.87,P<0.05).實時熒光定量PCR結果顯示,HepG2/ADM細胞中mdrl mRNA錶達水平較高,而HepG2/aqMDR、HepG2、HepG2/GFP三組細胞中mdrl的mRNA錶達水平差異沒有統計學意義(F＝2.30,P>0.05).結論 P-gP可以從耐藥肝癌細胞傳遞至敏感肝癌細胞,這一現象為進一步研究肝癌多藥耐藥機製提供瞭新的思路.
목적 탐토간암세포주HepG2세포간시부존재P-당단백(P-gp)적전체급P-gp여다약내약기인(mdrl)적관계.방법 장pSUPER.neo+GFP질립전염지HepG2세포(명명위HepG2/GFP),병여아매소내약HepG2세포(명명위HepG2/ADM)혼합배양.격광공취초현미경관찰간암세포간P-gp적전체.면역자주법분리혼합배양세포,수집원HepG2/GFP세포(명명위HepG2/aqMDR),채용Western blot분석HepG2、HepG2/ADM、HepG2/GFP,HepG2/aqMDR세포적P-gP표체수평,동시응용실시형광정량PCR분석저사세포mdrl mRNA적표체수평.다양본균수비교용단인소방차분석,진일보량량비교용SNK-q검험. 결과형광현미경하가견대량대록색형광적은정극륭.격광공취초현미경하견HepG2/aqMDR세포이황색형광위주,이HepG2/GFP이록색형광위주,HepG2/ADM이홍색형광위주.Western blot검측결과현시HepG2/aqMDR세포중P-gp표체수평저우HepG2/ADM,단명현고우HepG2/GFP(q＝35.07,P<0.05)여HepG2(q=36.87,P<0.05).실시형광정량PCR결과현시,HepG2/ADM세포중mdrl mRNA표체수평교고,이HepG2/aqMDR、HepG2、HepG2/GFP삼조세포중mdrl적mRNA표체수평차이몰유통계학의의(F＝2.30,P>0.05).결론 P-gP가이종내약간암세포전체지민감간암세포,저일현상위진일보연구간암다약내약궤제제공료신적사로.
Objective To investigate whether there is intercellular wansfer of functional P-glycoproteln (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 de-rived 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 mdrl 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). Coneinsions P-gp can transfer from drug resistant hepatocellular cells to sensitive hepatocellular carcimoma cells. This study suggests a novel mechanism of multidrng resistance inhepatocellular careimoma.
