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

[Abstract]ObjectiveTo 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.ResultsThe 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.ConclusionMACS 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.     

负载干细胞抗原的DC联合CIK细胞对乳腺癌荷瘤鼠肿瘤杀伤研究

目的：研究不同乳腺癌细胞抗原负载的DC-CIK细胞对于乳腺癌荷瘤鼠体内肿瘤的杀伤效果及其与Wnt通路的关系。方法：分离乳腺癌细胞系MCF-7/ADR中的干细胞并制备冻融抗原,以此冲击由正常人外周血提取的单个核细胞诱导培养的DC-CIK细胞,注入已建立的MCF-7/ADR乳腺癌荷瘤鼠模型中,并且与普通抗原冲击的DC-CIK细胞,未经抗原冲击DC-CIK细胞,单纯CIK细胞,及生理盐水组建立实验组及对照组,观察此5组中小鼠肿瘤细胞生长情况,通过原位末端标记法（TUNEL）检测各组肿瘤组织凋亡情况,免疫组化法检测bcl-2,Bax及β-catenin表达情况。结果：实验组和对照组小鼠肿瘤体积在治疗后存在差异,同组小鼠肿瘤体积体积在治疗前后也存在差异,对照组小鼠肿瘤体积最大（3.6245±0.09264）cm3,经干细胞抗原冲击的DC-CIK治疗组肿瘤体积最小（1.2342±0.13116） cm3,其tunel染色阳性率最高,bax表达阳性最强,bcl-2,β-catenin表达较其他组最弱。结论：经乳腺癌干细胞抗原冲击的DC-CIK细胞对乳腺癌荷瘤鼠的肿瘤组织较强的杀伤效果,其机制可能是Wnt/β-catenin信号通路中的β-catenin表达降低,并引起bax表达增高,bcl-2表达降低,从而引起肿瘤细胞凋亡。