CD133与胃癌细胞化疗耐药的关系及其机制

Relationship between CD133 and chemoresistance in human gastric cancer and its associated mechanism

目的探讨CDl33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、CDl33＋组及CDl33一组。Westernblot法和RT—PCR法分别检测分选后胃癌细胞CDl33、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CDl33表达后，检测CDl33、P-gP、Bcl-2、Akt及p-Akt蛋白及mRNA表达。结果CDl33＋组胃癌细胞中CDl33、P—gP及Bcl-2蛋白和mRNA表达水平均显著高于未分选组及CDl33-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CDl33＋组的抑制率显著低于CDl33一组（P〈0．05）。5-FU处理胃癌细胞后，CDl33＋组胃癌细胞中凋亡细胞比率明显低于CDl33-组及未分选组（P〈0．05）。CDl33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CDl33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，CDl33＋细胞可通过P13K／Akt通路起作用。

Objective To explore the relationship between CD133 ~ subsets ceils in human gastric cancer （GC） and molecules of drug resistance and their sensitivity to 5-FU. Methods Three gastric cancer cell lines therein KATO-m , 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 Hoeehst 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 ceils in CD133- subgroup as compared to other two subgroups, and the least apoptotie 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 invoiced in this process.