目的：探讨负载结直肠癌干细胞膜微粒（membrane microparticles,MMPs）DC-CIK 与西妥昔单抗（Cetuximab，C225）对EGFR靶向药耐药结直肠癌细胞的靶向协同杀伤作用及其机制。方法：PCR法检测结直肠癌SW480、SW620、HCT116株细胞k-RAS突变状态，无血清悬浮细胞培养法富集SW480、HCT116肿瘤干细胞，RT-PCR检测干细胞标志物Sox-2和Oct-4。提取外周血单个核细胞培养DC与CIK，将结直肠癌干细胞MMPs负载DC后再与CIK共孵育。形态观察及MTT法分别检测DC-CIK/CTL细胞及其培养上清、C225单独和合并处理对SW480、SW620、HCT116及其干细胞的杀伤效应；RT-PCR检测DC-CIK/CTL细胞培养上清、C225单独和合并作用对SW480、SW620、HCT116细胞凋亡相关基因Fas和上皮细胞间质转化（epithelial-mesenchymal transition, EMT）相关E-钙黏蛋白和波形蛋白基因的表达，划痕实验测定DC-CIK/CTL细胞培养上清、C225单独及联合作用对结直肠癌细胞侵袭行为影响。〖HT5W〗结果：〖HT5"SS〗SW480、SW620和HCT116细胞均为k-RAS突变型；富集培养获得的结直肠癌干细胞样细胞高表达Sox-2和Oct-4 mRNA。MMPs负载DC-CIK/CTL细胞及其分泌上清与C225对结直肠癌细胞的抑制有协同作用。C225与MMPs负载DC-CIK/CTL上清联合组相对于两者单独作用组能提高Fas与E-钙黏蛋白基因的表达；联合组的迁移率比两者单独作用组小。结论：负载结直肠癌干细胞MMPs的DC-CIK/CTL细胞对EGFR靶向药耐药结直肠癌细胞有体外特异靶向杀伤效应，且与C225显著协同，其发生机制与逆转细胞EMT和诱导细胞凋亡有关。

Objective: To explore killing effect of DC-CIK/CTL loaded on membrane microparticles (MMPs) of the colorectal cancer stem cells synergisted with cetuximab (C225) on colorectal cancer cells which are resistent to epidermal growth factor receptor (EGFR)-targeting drug C225, and their mechanisms. Methods: Mutation satus of k-RAS gene for colorectal cancer cell SW480, SW620 and HCT116 line cells were identified by PCR. Cancer stem cells of the SW480 and HCT116 were enriched with serum free suspension cell culture method. Stem cell markers, Sox-2 and Oct-4, were detected by RT-PCR. Pheripheral blood monocuclear cells were extracted to culture DC and CIK, CIK was co-cultured with DC loaded with MMP of the colorectal cancer stem cells. Observation of mophology and MTT assay were respectively used to detect killing effects of DC-CIK/CTL cells, their culture supernatant (DC-CIK/CTL’S) and C225 alone as well as both combination on the SW480, SW620, HCT116 line cells and their stem cells. Exprissions of apoptosis-related gene Fas, epithelial-mesenchymal transition (EMT)-related proteins, E-cadherin and vinmentin, in the SW480, SW620, HCT116 line cells after treatmented by DC-CIK/CTL’S and C225 alone as well as both combination were detected by RT-PCR. Effects of DC-CIK/CTL’S and C225 alone as well as both combination on invasion acts of the colorectal cance cells were detected by a scratch assay. Results: All of the SW480, SW620 and HCT116 cell lines were identified as K-RAS mutation type. Stem cells of the colorectal carcinoma which obtained from enrichment culture had a high expression of the stem cell marker Sox-2 and Oct-4. The DC-CIK/CTL cells loaded with MMPs, thier secretion supernatant and C225 had a synergistic effect on inhibition of colorectal cancer cells. the combination group of C225 with DC-CIK/CTL’S enhanced expressions of Fas and E-cadherin proteins, comparing with groups of C225 and DC-CIK/CTL’S alone. Migration rate of the cells in the combination treatment group was smaller than those in the single treatment groups. Conclusion: The DC-CIK/CTL loaded with the MMPs of colorectal cancer stem cell could be of specific targeting killing effect in vitro on the colorectal cancer cells which are resistant to EGFR-target drug. It could have a obvious synergistic effect with C225. Their mechanisms could be related to reverse of EMT and improvement of apoptosis of the cells.

国家自然科学基金资助项目（No. 31170880, 81371891)