槐耳颗粒和山药提取物联合DC-CIK细胞对结肠癌HT29细胞的体外抑制作用

Inhibitory effects of Huaier Granule or Chinese yam extract combined with DC-CIK cellson colon cancer HT29 cells in vitro

目的探讨槐耳颗粒和山药提取物联合树突状细胞-细胞因子诱导的杀伤细胞(DC-CIK细胞),对结肠癌HT29细胞的体外抑制作用。方法槐耳颗粒(500和1000 mg/L)、山药提取物(500和1000 mg/L)与结肠癌HT29细胞共孵育,观察细胞形态的变化,用四甲基偶氮唑盐(MTT)法检测药物对肿瘤细胞的增殖抑制情况,用流式细胞仪检测结肠癌HT29细胞中肿瘤干细胞(CD133+细胞)的比例变化。乳酸脱氢酶细胞毒性检测试剂盒检测DC-CIK细胞对药物作用前后的HT-29细胞的杀伤活性,流式细胞仪分析DC-CIK细胞作用后CD133+细胞的比例变化。结果槐耳颗粒(高剂量和低剂量)和山药提取物(高剂量和低剂量)均可抑制HT-29细胞的增殖能力(OD值分别为0.882±0.089,0.891±0.090,0.878±0.088,0.889±0.091),与空白对照组(1.086±0.103)相比,差异均有统计学意义(均P<0.05),而对比槐耳颗粒高、低剂量组和山药提取物高、低剂量组4个实验组间的抑制增殖能力显示,差异无统计学意义(P>0.05);与空白对照组相比,高、低剂量槐耳颗粒和山药提取物明显降低了HT29细胞中CD133+细胞比例(36.59±7.62,25.98±6.21,26.32±6.39,25.81±6.25,26.27±6.30),且差异有统计学意义(P<0.05),对比4个实验组间CD133+细胞比例,其差异无统计学意义(P>0.05);HT-29细胞经槐耳颗粒和高低剂量山药提取物处理后再经DC-CIK细胞杀伤,HT29细胞中CD133+细胞比例明显降低(17.01±5.89、16.58±5.79),差异有统计学意义(P<0.05)。细胞杀伤活性结果显示,随效靶比升高DC-CIK细胞对槐耳颗粒(27.32±2.65,32.45±2.78)和山药提取物(26.98±2.61,31.37±2.71)分别作用后的HT-29细胞的杀伤活性均有增强,效靶比相同时DC-CIK对肿瘤细胞的杀伤活性经槐耳颗粒(效靶比10∶1,21.02±2.06,27.32±2.65;效靶比20∶1,24.39±2.51,32.45±2.78)和山药提取物(效靶比10∶1,20.79±1.96,26.98±2.61;效靶比20∶1,24.11±2.32,31.37±2.71)作用后比作用前更强,差异均有统计学意义(均P<0.05)。结论槐耳颗粒和山药提取物对HT-29细胞具有明显抑制作用,且联合DC-CIK细胞后其体外抑制作用更强。

Objective To explore the inhibitory effects of Huaier Granule or Chinese yam extract combined with dendritic cells associated with cytokine-induced killers(DC-CIK)on colon cancer HT29 cells in vitro.Methods HT29 cells were treated with Huaier Granule(500 mg/L and 1000 mg/L)and Chinese yam extract(500 mg/L and 1000 mg/L).Morphology changes were observed.Methyl thiazolyl tetrazolium(MTT)method was used to detect the proliferation and inhibition of tumor cells.The change in proportion of cancer stem cells(CD133+cells)was detected by flow cytometry.The cytotoxicity of DC-CIK cells on HT-29 cells before and after treatment was detected by LDH cytotoxicity test kit,and the change in proportion of CD133+cells after treatment with DC-CIK cells was analyzed by flow cytometry.Results Huaier Granule(high dose and low dose)and Chinese yam extract(high dose and low dose)could inhibit the proliferation of HT-29 cells(0.882±0.089,0.891±0.090,0.878±0.088,0.889±0.091).Compared with the blank control group(1.086±0.103),The difference was statistically significant(P<0.05).But there were no statistically significant differences in inhibition effect between the four experimental groups(high dose and low dose Huaier Granule groups,high dose and low dose Chinese yam extract groups)(P>0.05).Compared with the blank control group,Huaier Granule(high dose and low dose)and Chinese yam extract(high dose and low dose)decreased the proportion of CD133+cells(36.59±7.62,25.98±6.21,26.32±6.39,25.81±6.25,26.27±6.30),and the differences were statistically significant(P<0.05),But there were no statistically significant differences inproportion of CD133+cells between the four experimental groups(P>0.05).The percentage of CD133+cells in HT-29 cells treated with Huaier Granule and Chinese yam extract decreased significantly after being treated with DC-CIK(17.01±5.89,16.58±5.79)and the differences were statistically significant(P<0.05).Cytotoxicity results showed that with an increasing ratio of effector cells to target cells,the killing activity of DC-CIK on HT29 cells after treated with Huaier Granule(27.32±2.65,32.45±2.78)or Chinese yam extract(26.98±2.61,31.37±2.71)showed an increasing trend.When ratio of effector cells to target cells was same,the killing activity of DC-CIK on HT29 treated with Huaier Granule(effect target ratio 10∶1,21.02±2.06,27.32±2.65;effect target ratio 20∶1,24.39±2.51,32.45±2.78)and Chinese yam extract(effect target ratio 10∶1,20.79±1.96,26.98±2.61;effect target ratio 20∶1,24.11±2.32,31.37±2.71)were higher than those before treatment,and the differences were statistically significant(P<0.05).Conclusion Huaier Granule and Chinese yam extract have obvious inhibitory effect on HT-29 cells,and the repression in vitro is stronger after combining with DC-CIK cells.