天津医药

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负载抗原DC与CIK共培养对富集培养乳腺癌CTCs杀伤作用研究

吕艳1,庞华2,司玉玲2,庞春淼2,孙雯雯2   

  1. 1. 天津医科大学研究生院
    2. 天津市第四中心医院肿瘤血液科及中心实验室
  • 收稿日期:2013-05-06 修回日期:2013-08-29 出版日期:2013-12-15 发布日期:2013-12-15
  • 通讯作者: 吕艳

Study on the Kill Activity of the Whole Tumor Cell Antigen Pulsed DC Co-Culture with CIK for Breast Cancer Circulating Tumor Cells

Lü Yan 1,PANG Hua 2,SI Yu ling3, PANG Chun miao3,SUN Wen wen3   

  1. 1. Graduate School of Tianjin Medical University
    2. Department of Tumor Hematology andCentral Laboratory, Tianjin Fourth Center Hospital
    3. Department of Tumor Hematology and Central Laboratory, Tianjin Forth Central Hospital
  • Received:2013-05-06 Revised:2013-08-29 Published:2013-12-15 Online:2013-12-15
  • Contact: Lü Yan

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摘要：

【摘要】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CK19mRNA条带表达，经体外培养后胞浆染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CD+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。

关键词: 树突细胞, 乳腺肿瘤, 细胞骨架, 角蛋白质类, 细胞凋亡, 循环肿瘤细胞, 表皮细胞黏附分子, 细胞因子诱导的杀伤细胞

Abstract:

[Abstract]   Objective  To study the kill activity of the whole tumor cell antigen (Ag) pulsed dendritic cell (DC) coculture 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 (CK8/18and 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 CK8/18and CK19of 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 CIK3groups, 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 typical 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 immune cells,which shows a high er proliferation and cytoxicty against breast cancer CTCs. This may be used as a clinically immunotherapy means of anti-breast cancer recurrence and metastasis.

Key words: dendritic cells, breast neoplasms, cytoskeleton, kerat, apoptosis, circulating tumor cells, epidermal cell adhesion molecule, 细胞因子诱导的杀伤细胞