CAJ | 학술논문

目的：观察负载抗原的树突状细胞（DC）与细胞因子诱导的杀伤细胞（CIK）对富集培养乳腺癌循环肿瘤细胞（CTCs）的细胞毒作用。方法将有CTCs的乳腺癌患者外周血单个核细胞，磁珠分选富集表皮细胞黏附分子（EpCAM）+乳腺癌CTCs体外培养，并用巢式PCR、细胞免疫荧光成像（CK8/18）、细胞免疫组化（CK8/18、CK19）方法进行鉴定，分选后EpCAM-细胞常规诱导DC及CIK；制备乳腺癌CTCs全冻融抗原（Ag）冲击DC，分Ag-DC-CIK组、DC-CIK组、DC组、CIK组。台盼蓝拒染法动态监测CIK细胞增殖情况，流式细胞术分析细胞免疫表型，MTT法检测对乳腺癌CTCs的杀伤活性，流式细胞术检测诱导乳腺癌CTCs的早期凋亡率，光镜及透射电镜观察细胞形态及超微结构。结果磁珠分选富集后EpCAM+细胞巢式PCR可见CK19 mRNA条带表达，经体外培养后胞浆染色CK8/18+、CK19+，CK-FITC/DAPI免疫荧光染色见胞浆蓝荧光/核绿荧光的CK8/18阳性细胞。DC与CIK共培养细胞增殖活性明显大于单独培养的CIK（P＜0.001），DC免疫表型CD1α、CD83+CD86+、CD83+CD11C+、CD86+CD11C+及CIK免疫表型CD3+CD8+、CD3+CD56+的表达率Ag-DC-CIK组高于DC-CIK组、DC组及CIK组（P＜0.05）。Ag-DC-CIK、DC-CIK、CIK3组均可诱导乳腺癌CTCs凋亡，凋亡率为（56.83±3.07）％、（31.43±1.77）％、（24.70±1.51）％，两两比较差异有统计学意义，透射电镜可见诱导凋亡乳腺癌CTCs的典型微结构。结论磁珠分选联合细胞免疫学方法可以提高乳腺癌CTCs的检测灵敏度；经抗原冲击DC明显增加CIK细胞的增殖能力和细胞毒活性,有可能作为一种临床有效的抗乳腺癌复发与转移的免疫治疗手段。
목적：관찰부재항원적수돌상세포（DC）여세포인자유도적살상세포（CIK）대부집배양유선암순배종류세포（CTCs）적세포독작용。방법장유CTCs적유선암환자외주혈단개핵세포，자주분선부집표피세포점부분자（EpCAM）+유선암CTCs체외배양，병용소식PCR、세포면역형광성상（CK8/18）、세포면역조화（CK8/18、CK19）방법진행감정，분선후EpCAM-세포상규유도DC급CIK；제비유선암CTCs전동융항원（Ag）충격DC，분Ag-DC-CIK조、DC-CIK조、DC조、CIK조。태반람거염법동태감측CIK세포증식정황，류식세포술분석세포면역표형，MTT법검측대유선암CTCs적살상활성，류식세포술검측유도유선암CTCs적조기조망솔，광경급투사전경관찰세포형태급초미결구。결과자주분선부집후EpCAM+세포소식PCR가견CK19 mRNA조대표체，경체외배양후포장염색CK8/18+、CK19+，CK-FITC/DAPI면역형광염색견포장람형광/핵록형광적CK8/18양성세포。DC여CIK공배양세포증식활성명현대우단독배양적CIK（P＜0.001），DC면역표형CD1α、CD83+CD86+、CD83+CD11C+、CD86+CD11C+급CIK면역표형CD3+CD8+、CD3+CD56+적표체솔Ag-DC-CIK조고우DC-CIK조、DC조급CIK조（P＜0.05）。Ag-DC-CIK、DC-CIK、CIK3조균가유도유선암CTCs조망，조망솔위（56.83±3.07）％、（31.43±1.77）％、（24.70±1.51）％，량량비교차이유통계학의의，투사전경가견유도조망유선암CTCs적전형미결구。결론자주분선연합세포면역학방법가이제고유선암CTCs적검측령민도；경항원충격DC명현증가CIK세포적증식능력화세포독활성,유가능작위일충림상유효적항유선암복발여전이적면역치료수단。
Objective To study the kill activity of the whole tumor cell antigen (Ag) pulsed dendritic cell (DC) co-culture with cytokine induced killer cell (CIK) for breast cancer circulating tumor cells (CTCs). Methods Peripheral blood mononuclear cells (PBMC) were isolated from breast cancer patients with CTCs using a blood cell separator instrument. The epidermal cell adhesion molecule (EpCAM ) (+) breast cancer CTCs enriched by MACS were cultured in vitro. The nested RT-PCR, cell immunofluorescence imaging (CK8/18), and immunohistochemistry (CK8/18 and CK19) methods, were used for the detection and identification of the cells. EpCAM (-) cells were routinely induced to DC and CIK, which were grouped into Ag-DC-CIK group, DC-CIK group, DC group and CIK group. The cytotoxic activity of co-cultured DC-CIK against breast cancer CTCs was detected by flow cytometry and MTT assay. The cell morphology was observed by light microscopy and transmission electron microscopy. Results The target band of CK19mRNA can be detected by nested RT-PCR. The expressions of CK8/18 and CK19 of EpCAM (+) cells in vitro were positive by immunofluorescence staining and immunohis-tochemical staining. The proliferative activity of co-cultured DC-CIK was higher than that of CIK ( P<0.001). The positive rates of CD1α+, CD83+CD86+, CD83+CD11C+, CD86+CD11C+DCs and CD3+CD8+, CD3+CD56+CIKs were significantly higher in Ag-DC-CIK group than those of DC-CIK group, DC group and CIK group (P<0.05). The apoptosis of breast cancer CTCs was induced in Ag-DC-CIK, DC-CIK and CIK3 groups, and apoptotic rates were (56.83±3.07)%, (31.43±1.77)%and (24.70±1.51)%, showing significant differences between them (P<0.05). Transmission electron microscopy showed the typi-cal micro-structure of breast cancer CTCs induced apoptosis. Conclusion MACS in combination with cell immunological methods can improve significantly the detective sensitivity of breast cancer CTCs. The co-cultured Ag-DC-CIK is highly effective im-mune cells,which shows a high er proliferation and cytoxicty against breast cancer CTCs. This may be used as a clinically immunother-apy means of anti-breast cancer recurrence and metastasis.