Li Gui-Xin, Zhao Shu-Shu, Zhang Xu-Guang, Wang Wen-Hao, Liu Jin, Xue Ke-Wei, Li Xiao-Yan, Guo Ying-Xue, Wang Li-Hua
Department of Oncology, Affiliated Hospital of Weifang Medical University, Weifang, Shandong 261031, P.R. China.
Department of Clinical Laboratory, Affiliated Hospital of Weifang Medical University, Weifang, Shandong 261031, P.R. China.
Mol Med Rep. 2015 Aug;12(2):2629-35. doi: 10.3892/mmr.2015.3765. Epub 2015 May 8.
The present study aimed to compare the antitumor effects of cascade primed immune (CAPRI) cells and cytokine-induced killer (CIK) cells in vitro, through investigating cell morphology, proliferation, cytotoxic activity to tumor cells and the ability of these cells to secrete cytokines. Peripheral blood samples (50 ml) were obtained from three healthy volunteers and peripheral blood mononuclear cells (PBMCs) were obtained from each via Ficoll-Conray density gradient centrifugation. Each suspension of PBMCs (1 x 10(6)/ml) was divided into two parts; CAPRI cells were obtained from one part through a series of induction, amplification and cytokine cultures, while CIK cells were obtained from the other part through induction with different cytokines. During the culture process, the proliferation and morphological changes were observed for the two cell types using Trypan blue staining. At day 14, the cytotoxic activity of the two cell types was examined through determining lactate dehydrogenase release in the presence of K562 leukemia cells and MCF-7 breast cancer cells. In addition, secretory levels of interferon (IFN)-γ and interleukin (IL)-2 were detected using enzyme-linked immunospot (ELISPOT) technology. The results revealed that at day 5 and 14 of culture, there were significantly fewer CAPRI cells compared with CIK cells (P<0.001), although the survival rate of each cell type was >95%. The cytotoxic activity of CAPRI cells towards the K562 cell line was effector-target ratio-dependent (40:1 and 20:1) with values of 55.1 ± 3.25 and 35.0 ± 2.65%, respectively, which were significantly reduced compared with the corresponding data in CIK cells, 60.0 ± 3.03 and 39.7 ± 3.42% (P=0.004 and 0.005, respectively). Furthermore, the cytotoxic activity of CAPRI cells towards MCF-7 cells were 71.5 ± 3.06, 56.0 ± 3.76 and 40.2 ± 2.90% at effector-target ratios 40:1, 20:1 and 10:1, respectively. These data were significantly higher than the corresponding values in CIK cells, 65.4 ± 3.86, 49.5 ± 3.91 and 36.1 ± 3.73% (P=0.002, 0.003 and 0.02, respectively). As determined using ELISPOT technology at different cell concentrations (1 x 10(6)/ml and 5 x 10(5)/ml), IFN-γ secretion levels, determined by the number of spot-forming cells, of CAPRI cells were 126.2 ± 10.31 and 48.8 ± 10.99, respectively, which were significantly reduced compared with those of CIK cells, 409.3 ± 7.76 and 159.3 ± 15.45, respectively (P<0.001). IL-2 secretion levels in CAPRI cells were 325.1 ± 16.24 and 113.8 ± 11.29 at 1 x 10(6)/ml and 5 x 10(5)/ml, respectively, which were significantly increased compared with CIK cells, 212.0 ± 16.58 and 70.7 ± 10.57, respectively (P<0.001). In conclusion, the present study demonstrated that CAPRI cells had a reduced proliferation rate compared with CIK cells as well as a less potent cytotoxic effect on K562 cells; however, the two cell types had potent cytotoxic activity towards solid tumor MCF-7 cells. In addition, CAPRI cells secreted lower levels of IFN-γ and increased levels of IL-2 compared with CIK cells. These results indicated that antitumor activities of CAPRI and CIK cells proceeded via different mechanisms.
本研究旨在通过研究细胞形态、增殖、对肿瘤细胞的细胞毒性活性以及这些细胞分泌细胞因子的能力，比较级联启动免疫（CAPRI）细胞和细胞因子诱导的杀伤（CIK）细胞在体外的抗肿瘤作用。从三名健康志愿者获取外周血样本（50 ml），并通过Ficoll-Conray密度梯度离心从每份样本中获得外周血单个核细胞（PBMC）。每份PBMC悬液（1×10⁶/ml）分为两部分；通过一系列诱导培养、扩增和细胞因子培养从一部分获得CAPRI细胞，而通过用不同细胞因子诱导从另一部分获得CIK细胞。在培养过程中，使用台盼蓝染色观察两种细胞类型的增殖和形态变化。在第14天，通过测定存在K562白血病细胞和MCF-7乳腺癌细胞时乳酸脱氢酶的释放来检测两种细胞类型的细胞毒性活性。此外，使用酶联免疫斑点（ELISPOT）技术检测干扰素（IFN）-γ和白细胞介素（IL）-2的分泌水平。结果显示，在培养的第5天和第14天，与CIK细胞相比，CAPRI细胞数量显著减少（P<0.001），尽管每种细胞类型的存活率均>95%。CAPRI细胞对K562细胞系的细胞毒性活性呈效应细胞与靶细胞比例依赖性（40:1和20:1），值分别为55.1±3.25%和35.0±2.65%，与CIK细胞中的相应数据60.0±3.03%和39.7±3.42%相比显著降低（分别为P=0.004和0.005）。此外，CAPRI细胞对MCF-7细胞的细胞毒性活性在效应细胞与靶细胞比例为40:1、20:1和10:1时分别为71.5±3.06%、56.0±3.76%和40.2±2.90%。这些数据显著高于CIK细胞中的相应值，分别为65.4±3.86%、49.5±3.91%和36.1±3.73%（分别为P=0.002、0.003和0.02）。使用ELISPOT技术在不同细胞浓度（1×10⁶/ml和5×10⁵/ml）下测定，由斑点形成细胞数量确定的CAPRI细胞的IFN-γ分泌水平分别为126.2±10.31和48.8±10.99，与CIK细胞的分泌水平相比显著降低，CIK细胞的分泌水平分别为409.3±7.76和159.3±15.45（P<0.001）。在1×10⁶/ml和5×10⁵/ml时，CAPRI细胞中IL-2的分泌水平分别为325.1±16.24和113.8±11.29，与CIK细胞相比显著升高，CIK细胞的分泌水平分别为212.0±16.58和70.7±10.57（P<0.001）。总之，本研究表明，与CIK细胞相比，CAPRI细胞的增殖率降低，对K562细胞的细胞毒性作用较弱；然而，这两种细胞类型对实体瘤MCF-7细胞具有较强的细胞毒性活性。此外，与CIK细胞相比，CAPRI细胞分泌较低水平的IFN-γ和较高水平的IL-2。这些结果表明，CAPRI细胞和CIK细胞的抗肿瘤活性通过不同机制发挥作用。
