目的 探讨CD133阳性胃癌起始细胞对常规化疗药物氟尿嘧啶(5-FU)的敏感性及其耐药机制.方法 免疫磁珠法分选KATO-Ⅲ、SGC7901和MKN-45胃癌细胞株并分为未分选组、CD133+组及CD133-组.Western blot法和RT-PCR法分别检测分选后胃癌细胞CD133、P-gp、Bax和Bcl-2蛋白及mRNA表达水平.免疫荧光法检测各组细胞P-gp及Bcl-2蛋白的表达.CCK-8法和Hoechest染色检测各组细胞对5-FU的敏感性.siRNA干扰MKN45细胞CD133表达后,检测CD133、P-gp、Bcl-2、Akt及p-Akt蛋白及mRNA表达.结果 CD133+组胃癌细胞中CD133、P-gp及Bcl-2蛋白和mRNA表达水平均显著高于未分选组及CD133-组(均P＜0.05),而促凋亡因子Bax的表达水平则明显降低(P＜0.05).在相同药物浓度下,5-FU对CD133+组的抑制率显著低于CD133-组(P＜0.05).5-FU处理胃癌细胞后,CD133+组胃癌细胞中凋亡细胞比率明显低于CD133-组及未分选组(P＜0.05).CD133特异siRNA干扰胃癌细胞后,干扰组P-gp、Bcl-2和p-Akt蛋白及mRNA表达显著降低(均P＜0.05),而Bax表达水平则显著增加(P＜0.05).结论 CD133可能通过调节P-gp和Bcl-2的表达来促进胃癌的化疗耐药；在胃癌化疗耐药产生中,CD133+细胞可通过PI3K/Akt通路起作用.%Objective To explore the relationship between CD133 + subsets cells in human gastric cancer (GC) and molecules of drug resistance and their sensitivity to 5-FU.Methods Three gastriccancer cell lines therein KATO-Ⅲ,SGC7901 and MKN45 were sorted by immunomagnetic beads cell sorting method.Then above cell lines were further divided into un-sorted GC cells,CD133+ subgroup and CD133-subgroup.The expressions of CD133,P-gp,Bax and Bcl-2 were determined by RT-PCR,Western blot and immunoflurescence.Meanwhile,the sensitivity to 5-FU of three subgroups was detected by CCK-8 Kit.The apoptosis induced by 5-FU in three subgroups was determined by Hoechst 33258.Results Expressions of CD133 in three CD133+ subgroups were significantly higher than those in un-sorted GC cells and CD133-subgroup (all P＜0.05).Expressions of P-gp and Bcl-2 in the three GC cell lines were different (all P＜0.05).There were significant differences of expressions of P-gp,Bcl-2 and Bax among CD133+ cells,un-sorted GC cells and CD133-cells (all P＜0.05).CCK-8 detection showed that CD133-subgroup of MKN45 GC cell line was more sensitive than CD133+ cells to 5-FU (P＜0.05).Hoechst 33258 staining showed that there were more apoptotic cells in CD133-subgroup as compared to other two subgroups,and the least apoptotic cells were observed in CD133+ subgroup of MKN45 GC cell line (P＜0.05).CD133 siRNA was transfected into MKN45 GC cell line and could down-regulate the expressions of CD133,P-gp,Bcl-2 and p-Akt,while the expression of Bax increased (all P＜0.05).Conclusions CD133 may contribute to the resistance of GC cells to chemotherapy drug through P-gp,Bcl-2 and Bax.PI3K/Akt signal pathway may be involved in this process.
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