牛膝多糖刺激的DC联合CIK细胞对SW480的杀伤作用研究

Effect of Achyranthes bidentata polysaccharides stimulated dendritic cells co-cultured with cytokine induced killer cells against SW480 cells

从中药材牛膝中提取牛膝多糖(ABPS),用ABPS和/或SW480肿瘤抗原刺激的树突状细胞(DC)联合细胞因子诱导的杀伤性细胞(CIK)对结肠癌细胞株SW480进行杀伤试验,研究ABPS刺激的DC联合CIK细胞的抗肿瘤效果。分离人外周血单个核细胞培养成DC和CIK细胞。①把DC分成4个组合:单纯DC组作为对照组,ABPS刺激DC实验组(ABPS刺激的质量浓度为50mg·L-1),SW480肿瘤抗原刺激DC实验组,ABPS(50mg·L-1)和SW480肿瘤抗原联合刺激DC实验组,流式检测比较各组DC细胞表面分子CD80,CD86,CD11c,CD40,HLA-DR的阳性率的差异(每组均为6例样本)。②以上述4个组合的DC与CIK细胞以1:5比例混合共同作为效应细胞;以SW480肿瘤细胞株为靶细胞,设置效靶比分别为30:1,20:1及10:1,进行细胞杀伤活性试验,用CCK-8试剂盒检测、比较各组细胞杀伤活性的差异。③ELISA检测并比较上述细胞杀伤活性试验中效靶比为30:1的实验组各反应孔(3个复孔)上清液中细胞因子IL-2,IL-12p70,IL-17及TNF-α的分泌水平的差异。结果显示:①ABPS和SW480肿瘤抗原联合刺激的DC表面CD80,CD11c,HLA-DR的阳性率显著高于其他实验组的DC(P<0.05);CD86,CD40的阳性率显著高于单纯DC实验组(P<0.05),与其他实验组无显著差异。②在效靶比分别为30:1,20:1及10:1细胞杀伤活性试验中,ABPS刺激的DC+CIK细胞组、SW480肿瘤抗原刺激的DC+CIK细胞组对SW480肿瘤细胞株的杀伤活性显著高于DC+CIK细胞组(P<0.05);ABPS+SW480肿瘤抗原+DC+CIK实验组对SW480肿瘤细胞株的杀伤活性均显著高于其他实验组(P<0.05)。③效靶比为30:1的细胞杀伤活性试验中,ABPS+SW480抗原+DC+CIK细胞组上清液中IL-12p70,TNF-α的分泌水平高于其他实验组,差异具有统计学意义(P<0.05);IL-2,IL-17的分泌水平各组之间无显著差异。说明ABPS刺激的DC+CIK细胞对SW480肿瘤细胞株的杀伤活性显著提高,ABPS和SW480肿瘤抗原共同刺激DC能协同CIK提高对SW480肿瘤细胞株的杀伤活性。

Achyranthes bidentata polysaccharides （ABPS） was extracted from the root of A. bidentata. Dendritic cells （DC） , which were stimulated with ABPS and/or tumor antigen SW480, were cocultured with cytokine induced killer cells （CIK） to test the cytotoxic effect on colon cancer cell line SW480. Peripheral bloodmononuclear cells （PBMNCs） which were separated from human peripheral blood were cultured to DC and CIK separately. ①DC were divided into four groups： pure DC served as control group; ABPS （50 mg · L- l ） stimulated DC served as experimental group; SW480 tumor antigen stimulated DC served as the second experimental group; ABPS（50 mg · L-1） and SW480 tumor antigen co-stimulated DC served as the third experimental group. Flow cytometry was used to detect the difference of the positive rate of molecules in the cell surface of DC, include CD80, CD86, CD1 lc, CD40, HLA-DR （6 samples for each group）. ②The four DC groups were mixed with CIK at the ratio 1：5 and acted as effect cells （ DC ＋ CIK groups） , and the colon cancer cell line SW480 acted as target cells. The effect cells and the target cells were mixed together at the ratio 30： 1, 20：1 and 10：1 separately, and the CCK-8 kit was used to test the cytotoxic effect on colon cancer cell line SW480. ③ At the mixing ratio 30：1 of effect cells and target cells, ELISA was used to test the level of cytokines secretion, including 1L-2, IL-12p70, IL-17 and TNF-cl, in the liquid snpernatant of every test group （3 duplication per sample）. The results showed as following： （~） The positive rates of CD80, CDllc, HLA-DR, in the cell surface of DC which was co-stimulated by ABPS （50 mg · L -1 ） and SW480 tumor antigen, were obviously higher than the other DC groups （P 〈 0. 05） , and the positive rates of CD86, CD40 were obviously higher than the pure DC group （P 〈0. 05） , and there was no remarkable difference with the other two DC groups. ②At the mixing ratio 30： 1, 20：1 and 10：1 of the effect cells and the target cells, the cytotoxic effect of ABPS stimulated DC ＋ CIK group and SW480 tumor antigen stimulated DC ＋ CIK group was obviously higher than DC ＋ CIK group （ P 〈 0. 05 ） , the cytotoxic effect of ABPS and SW480 tumor an tigen co-stimulated DC ＋ CIK group was obviously higher than all the other groups. ③At the mixing ratio 30：1 of the effect cells and the target cells, the secretion levels of IL-12p70 and TNF-α in the liquid supernatant of the ABPS and SW480 tumor antigen co-stimulated DC ＋ CIK group were obviously higher than all the other groups （P 〈0. 05）, the secretion levels of IL-2 and IL-17 in the liquid supernatant of every test group have no remarkable difference. The cytotoxic effect of ABPS stimulated DC ＋ CIK on SW480 was obviously increased. The cytotoxic effect of ABPS and SW480 tumor antigen co-stimulated DC ＋ CIK group was obviously higher than all the other.