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[摘要]
该文探讨了毛酸浆果提取物(Physalis pubescens L. Fruits Extract,PPFE)对RAW264.7巨噬细胞免疫调节作用。采用噻唑蓝比色法(MTT)确定细胞毒性和增殖活性,格里斯法(Griess)、酶联免疫法(ELISA)、荧光探针法、中性红吞噬实验测定体外培养的巨噬细胞中一氧化氮(NO)、肿瘤因子-α(TNF-α)和白介素-6(IL-6)、干扰素-γ(IFN-γ)、活性氧(ROS)含量以及吞噬率;流式细胞术测定细胞周期变化及细胞凋亡情况,荧光定量反转录-聚合酶链反应(qRT-PCR)检测TNF-α、IFN-γ以及IL-6等细胞因子mRNA表达水平。结果显示:运用UHPLC-Q-TOF-MS测定PPFE化学成分分析,共鉴定出14种黄酮类物质。PPFE作用RAW264.7细胞的安全浓度在25~250 μg/mL范围内,与空白组相比,毛酸浆果提取物质量浓度为25 μg/mL时免疫增强效果最佳,其细胞增殖率、NO释放量、吞噬率、相对活性氧水平、TNF-α、IFN-γ、IL-6的分泌量分别为130.53%、27.79 μmol/L、189.88%、137.75%、150.54 pg/mL、119.36 pg/mL、15.41 pg/mL。PPFE治疗组的细胞周期的G0/G1期、S期、G2/M期百分比分别为53.08%、17.40%、29.52%。细胞凋亡率与空白组相比降低了52.80%,同时极显著(p<0.01)上调TNF-α、IFN-γ、IL-6的表达。综上可知,PPFE通过调节RAW264.7细胞吞噬功能、分泌免疫因子、细胞周期分布及凋亡情况,以及上调细胞因子mRNA表达水平,增强RAW264.7细胞免疫调节作用。
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[Abstract]
To investigate the immunomodulatory effect of Physalis pubescens L. fruit extract (PPFE) in RAW264.7 cells, the cytotoxicity and proliferative activity were determined by the thiazole blue colorimetric method (MTT). Griess assay, enzyme-linked immunosorbent assay (ELISA), fluorescent probe technique and neutral red phagocytosis assay were used to measure the contents of nitric oxide (NO), tumor factor-α (TNF-α), IFN-γ, IL-6 and reactive oxygen species (ROS) as well as the phagocytosis rate. The changes in cell cycle and apoptosis were measured by flow cytometry, and the mRNA expression levels of cytokines such as TNF-α, IFN-γ and IL-6 were determined by fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The results showed that UHPLC-Q-TOF-MS was used to determine the chemical composition of PPFE, and a total of 14 flavonoids were identified. The safe concentration of PPFE for RAW264.7 cells was in the range of 25~250 μg/mL. Compared with the blank group, the immunoenhancing effect was the strongest at 25 μg/mL, with the corresponding cell proliferation rate, content of released NO, phagocytosis rate, relative ROS level, and contents of secreted TNF-α, IFN-γ and IL-6 were 130.53%, 27.79 μmol/L, 189.88%, 137.75%, 150.54 pg/mL, 119.36 pg/mL, 15.41 pg/mL, respectively. The percentages of G0/G1 phase, S phase and G2/M phase of the cell cycle were 53.08%, 17.40%, and 29.52%, respectively. Compared with the blank group, the apoptosis rate was reduced by 52.80%, while the expressions of TNF-α, IFN-γ and IL-6 were significantly up-regulated (p<0.01) for the PPFE treated group. In summary, PPFE can enhance the immunomodulatory effect of RAW264 cells by regulating the phagocytosis, secretion of immune factors, cell cycle distribution and apoptosis, as well as up-regulating the mRNA expression levels of cytokines.
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