摘要
通过体外细胞试验,探讨蔓菁粗多糖对RAW264.7 巨噬细胞的免疫活性影响.取对数生长期的巨噬细胞,调整细胞含量至1.2 × 105个/mL,接种于96孔板培养24 h,分别设置阴性对照(只加完全培养基),阳性对照组(LPS,1μg/mL),蔓菁粗多糖组(50,100,200,400 μg/mL),培养24 h后MTT法检测细胞增殖活力、吞噬能力、分泌NO能力;小鼠颈椎脱臼处死,无菌环境下取脾脏,调整细胞含量为1.0 ×106个/mL,接种于96孔板,设对照组、蔓菁粗多糖组(50,100,200,400 μg/mL)以及Con A(10 μg/mL)阳性对照组或LPS(5 μg/mL)阳性对照组,培养24,48,72 h.培养结束前4h,每孔加入MTT(5 mg/mL)10 μL,离心弃上清液,加入100 μL DMSO充分振荡后,用酶标仪测定波长570 nm处的吸光度,计算增殖指数.与阴性对照组相比,蔓菁粗多糖组(50 μg/mL)巨噬细胞存活率显著升高(p=0.001＜0.01),LPS组、蔓菁粗多糖组(200,400 μg/mL)巨噬细胞存活率升高(p=0.011,0.021,0.027＜0.05);与阴性对照组相比,LPS组与蔓菁粗多糖各剂量组巨噬细胞吞噬指数显著升高(p＜0.01),与LPS组相比,蔓菁粗多糖组(50,100,200 μg/mL)巨噬细胞吞噬指数显著升高(p＜0.01),蔓菁粗多糖组(400 μg/mL)巨噬细胞吞噬指数升高(p＜0.05);与阴性对照组相比,LPS组及蔓菁粗多糖组(50 μg/mL)NO表达量显著升高(p＜0.01),与LPS组相比,蔓菁粗多糖组(200 μg/mL)NO表达量显著降低(p＜0.01),蔓菁粗多糖组(100,400 μg/mL)NO表达量降低(p＜0.05);与阴性对照组相比,蔓菁粗多糖组(50,100,200 μg/mL)脾细胞(T细胞)增殖指数显著升高(p＜0.01),conA组脾细胞(T细胞)增殖指数升高(p＜0.05),与conA组相比,蔓菁粗多糖组(200 μg/mL)脾细胞(T细胞)增殖指数显著升高(p＜0.01),蔓菁粗多糖组(50 μg/mL)脾细胞(T细胞)增殖指数升高(p＜0.05);与阴性对照组相比,蔓菁粗多糖组(100,200,400 μg/mL)脾细胞(B细胞)增殖指数显著升高(p＜0.01),LPS组及蔓菁粗多糖组(50 μg/mL)脾细胞(B细胞)增殖指数升高(p＜0.05),与LPS组相比蔓菁粗多糖组(200,400 μg/mLl)脾细胞(B细胞)增殖指数升高(p＜0.05).蔓菁粗多糖对RAW264.7细胞具有很好的免疫调节活性,蔓菁粗多糖对小鼠细胞免疫及体液免疫均有较强的促进作用.
Abstract
Exploring the immune activity of RAW264.7 Macrophages affected by extracellular polysaccharides from Manjing through in vitro cell experiments.Took logarithmic growth stage macrophages and adjust the cell concentration to 1.2 × 105 cells/mL,inoculate on a 96 well plate for 24 h,and set up a negative control group(only complete culture medium added)and a positive control group(LPS,1 μg/mL),the crude polysaccharide group of Manjing(50,100,200,400 μg/mL),after 24 h of cultivation,MTT assay was used to detect cell proliferation activity,phagocytic ability,and NO secretion ability;Mouse cervical dislocation was euthanized,and the spleen was taken in a sterile environment.The cell concentration was adjusted to 1.0 × 106 mL and inoculated onto a 96 well plate,set up a control group and a crude polysaccharide group(50,100,200,400 μg/mL)and Con A(10 μg/mL)positive control group or LPS(5 μg/mL)positive control group,cultivate for 24,48,72 h.4 h before the end of cultivation,add MTT(5 mg/mL)10 μL to each well.Centrifuge the supernatant and sufficient shake after adding 100 μL DMSO,measure the absorbance at 570 nm using an enzyme-linked immunosorbent assay(ELISA)reader and calculate the proliferation index.Compared with the negative control group,the group treated with crude polysaccharides from Manjing(50 μg/mL),the survival rate of macrophages significantly increased(p=0.001＜0.01),LPS group,Manjing crude polysaccharide group(200,400 μg/mL)the survival rate of macrophages increased(p=0.011,0.021,0.027＜0.05);Compared with the negative control group,the phagocytic index of macrophages in the LPS group and the various dose groups of crude polysaccharides from Primulaceae significantly increased(p＜0.01),compared with the LPS group,the crude polysaccharide group of Manjing crude polysacchande group(50,100,200μg/mL),the phagocytic index of macrophages significantly increased(p＜0.01),Manjing crude polysaccharide group(400μg/mL),the phagocytic index of macrophages increased(p＜0.05);Compared with the negative control group,the LPS group and the crude polysaccharide group of Manjing crude polysacchande group(50 μg/mL),the expression level of NO significantly increased(p＜0.01),compared with the LPS group,the crude polysaccharide group of Manjing(200 μg/mL)the expression level of NO significantly decreased(p＜0.01),Manjing crude polysaccharide group(100,400 μg/mL)the expression level of NO significantly decreased(p＜0.05);Compared with the negative control group,the group treated with crude polysaccharides from Manjing(50,100,200 μg/mL),the proliferation index of spleen cells(T cells)significantly increased(p＜0.01),the proliferation index of splenocytes(T cells)in the conA group increased(p＜0.05),compared with the conA group,the crude polysaccharide group of Manjing(200 μg/mL),the proliferation index of spleen cells(T cells)significantly increased(p＜0.01),while the group treated with crude polysaccharides from Primulaceae showed a significant increase(50 μg/mL),the proliferation index of spleen cells(T cells)increased(p＜0.05);Compared with the negative control group,the crude polysaccharide group of Manjing(100,200,400 μg/mL),the proliferation index of spleen cells(B cells)significantly increased(p＜0.01),LPS group and crude polysaccharides group of Manjing crude polysacchande group(50％ μg/mL),the proliferation index of spleen cells(B cells)increased(p＜0.05),compared with the LPS group,the crude polysaccharide group of Manjing(200,400 μg/mL),the proliferation index of spleen cells(B cells)increased(p＜0.05).Manjing crude polysaccharide had good immunomodulatory activity on RAW264.7 cells;Manjing crude polysaccharide had a strong promoting effect on both cellular and humoral immunity in mice.
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