斗米虫蛋白体外抗肿瘤活性及其对小鼠巨噬细胞免疫调节作用

Anti-Tumor Activity of the Protein of Apriona swainsoni Larvae In Vitro and Its Immunoregulatory Effect on Mouse Macrophages

该文主要为了研究斗米虫蛋白的体外抗肿瘤活性及其免疫调节作用。提取斗米虫总蛋白,逐级盐析分为3个部分,透析除盐后得到不同蛋白部位,并采用SDS-PAGE检测斗米虫不同蛋白部位的分子量;利用MTT法、流式细胞术等方法研究斗米虫蛋白对人胃癌细胞MFC和小鼠乳腺癌细胞4T1增殖、迁移和凋亡的作用。MTT法研究斗米虫蛋白对小鼠单核巨噬细胞RAW264.7和人脐静脉内皮细胞HUEVC增殖的影响;荧光微球吞噬实验检测斗米虫蛋白对RAW264.7细胞吞噬能力的影响;Griess法检测对RAW264.7细胞释放NO能力的影响;ELISA法检测对RAW264.7细胞的IL-6、TNF-α和IL-1β分泌量;RT-PCR法检测不同浓度的斗米虫蛋白作用后RAW264.7细胞中TNF-α、IL-1、TLR4、MIR-7、IFN-γ、TRL-4、IL-6以及4T1细胞中MMP2、MMP9、STAT3、c-Myc和Sdf1 mRNA水平变化。结果显示,斗米虫蛋白主要分子量集中于63 kDa,斗米虫蛋白对人胃癌细胞MFC及小鼠乳腺癌细胞4T1的增殖表现出较好抑制作用,并呈现出一定剂量依赖关系(P<0.05),对HUEVC细胞没有细胞毒性,对RAW264.7细胞表现出较好的促进增殖的作用(P<0.05)。斗米虫蛋白实验组与正常组细胞相比可以抑制4T1细胞的迁移(P<0.01),可诱导MFC和4T1细胞凋亡(P<0.05);斗米虫蛋白能够提高RAW264.7细胞的吞噬活性、NO释放量、TNF-α、1L-1β和IL-6分泌量及TNF-α、IL-1、TLR4、MIR-7、IFN-γ和IL-6细胞因子的mRNA水平以及能显著下调4T1细胞中MMP2、MMP9、STAT3、c-Myc和Sdf1 mRNA水平(P<0.05,P<0.01)。由此推论,斗米虫蛋白具有较好的体外抗肿瘤活性并且具有潜在的免疫调节作用。

Here,we studied the anti-tumor activity and immunomodulatory effects of the protein of Apriona swainsoni larvae in vitro.The total protein of Aprionas wainsoni larvae was extracted and divided into three parts.The different protein sites were obtained after dialysis and salt removal.The molecular weights of different proteins of Apriona swainsoni larvae were detected by SDS-PAGE.The MTT method was used to study the human gastric cancer MFC,the mouse breast cancer 4T1 cells,macrophage RAW264.7 cells and the human umbilical endothelial vein HUEVC cells proliferation.Migration assay was used to detect the effects of the protein of Apriona swainsoni larvae on the migration ability of 4T1 cells.Flow cytometry was used to study the effect of the protein of Apriona swainsoni larvae on apoptosis of MFC,4T1 cells.Fluorescence microspheres assay for the effect of the the protein of Apriona swainsoni larvae on phagocytic ability of RAW264.7 cells.The production of NO(nitric oxide)production wasevaluated by Griess.The production of TNF-α(tumor necrosis factor-α),IL-6(interleukin-6),and IL-1(interleukin-1)were determined by ELISA(immunosorbent assay).The mRNA levels of TNF-α(tumor necrosis factor-α),IL-1(interleukin-1),TLR4(toll-like receptors 4),MIR-7(mirn-7),IFN-γ(interferon-γ),IL-6(interleukin-6)associated with cellphagocytic ability in RAW264.7 cells were detected by RT-PCR.The mRNA levels of MMP2(matrix metalloprotein 2),MMP9(matrix metalloprotein 9),signal transducer and STAT3(signal transducer and activators of transcription 3),c-Myc and Sdf1(stromal cell-derived factor 1)associated with cell proliferation and migration in 4T1 cells were detected by RT-PCR.Compared with the normal group,the protein of Apriona swainsoni larvae showed a good inhibitory effect on the proliferation of MFC and 4T1 cells,and had no effect on the proliferation of RAW264.7 cells(P<0.05).The cell migration rates of test groups were reduced(P<0.01)when compared with the normal group,suggesting that the protein of Apriona swainsoni larvae inhibited the migration of 4T1 cells.The protein of Apriona swainsoni larvae could induce apoptosis in MFC and 4T1 cells(P<0.05),and have no cytotoxic effect on HUEVC cells.The protein of Apriona swainsoni larvae significantly increased phagocytic ability and production of NO,TNF-α,IL-6 and IL-1β.The mRNA levels of TNF-α,IL-1,TLR4,MIR-7,IFN-γand IL-6 in RAW264.7 cells of the test groups were significantly upregulated(P<0.05,P<0.01).The mRNA levels of MMP2,MMP9,STAT3,c-Myc and Sdf1 in 4T1 cells of the test groups were significantly upregulated(P<0.05,P<0.01).The protein of Apriona swainsoni larvae has good in vitro antitumor activity and potential immunomodulatory effects.