资木瓜总苷抑制体外培养的原代小鼠骨髓源性肥大细胞脱颗粒  

Total saponins of Chaenomeles speciosa inhibit the degranulation of primary mouse bone marrow-derived mast cells in vitro

作　　者：贾云莉 李世刚[1] 柳蔚[1] 喻玲玲[1] 曾小威[1] 

机构地区：[1]三峡大学医学院,湖北宜昌443002

出　　处：《细胞与分子免疫学杂志》2015年第10期1350-1353,1357,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81274166)

摘　　要：目的研究资木瓜总苷对小鼠骨髓来源的肥大细胞(BMMC)脱颗粒影响。方法从C57BL/6小鼠股骨获得骨髓细胞,用含100 m L/L胎牛血清、20 ng/m L IL-3和40 ng/m L干细胞因子(SCF)的RPMI1640培养基,培养4周;流式细胞术检测CD117、FcεRIα双阳性细胞的比例,甲苯胺蓝染色法观察细胞的成熟度。资木瓜总苷与诱导成功的BMMC共培养,CCK-8法检测资木瓜总苷对BMMC的毒性作用,荧光分光光度法定量检测β-己糖胺酶释放量,并计算其释放率,ELISA检测类胰蛋白酶释放量。结果原代培养诱导4周后,CD117和FcεRIα双阳性细胞比例大于95%,且核被染蓝色,胞质为紫红色,呈现肥大细胞特性。(0.01、0.03、0.10)mg/m L的资木瓜总苷作用12 h,对BMMC无毒性作用。与空白对照组相比,资木瓜总苷与交联牛血清白蛋白的二硝基苯半抗原(DNP-BSA)、A23187刺激的BMMC作用后,细胞的β-己糖胺酶释放率和类胰蛋白酶的释放量均显著降低。结论资木瓜总苷对不同抗原刺激原代培养的小鼠BMMC脱颗粒具有显著抑制作用,且存在量效关系。Objective To study the effects of total saponins of Chaenomeles speciosa on the degranulation of mouse bone marrow-derived mast cells （BMMCs）. Methods Bone marrow cells were isolated from C57BL/6 mice and were cultured in RPMI1640 medium containing 100 mL/L fetal bovine serum, 20 ng/mL IL-3 and 40 ng/mL stem cell factor （SCF）. After four-week culture, flow cytometry was used to identify the purity of double-positive （CDl17 · FCERIα ） mast cells; toluidine blue was used to detect the maturity of mast cells. After total saponins of Chaenomeles speciosa was added into the medium of BMMCs, CCK-8 assay was performed to assess the toxic effects of total saponins of Chaenomeles speciosa on BMMCs; the amount of β-hexosaminidase release was detected by fluorescence spectrophotometry; the content of tryptase in cell supernatants was detected by ELISA. Results After cultured for four weeks, the purity of double-positive cells was more than 95%, and the cells presented the features of mature mast cells, with blue nuclear and purple cytoplasm by toluidine blue staining. After BMMCs were cultured in the presence of total saponins of Chaenomeles speciosa （0.01, 0. 03, 0.10 mg/mL） for 12 hours, CCK-8 assay indicated that the total saponins did not exert the toxic effects on the BMMCs. Fluorescence spectrophotometry revealed that the release amount and release rate of β-hexosaminidase decreased in the cells treated with the total saponins of Chaenomeles speciosa, DNP-BSA and A23187 compared with the controls, and ELISA assay showed that the amount of tryptase release in the supernatants was reduced as well. Conclusion Total saponins of Chaenomeles speciosa can inhibit the degranulation of primary mouse BMMCs stimulated by different antigens with a clear dose-effect relationship.

关 键 词：资木瓜总苷 骨髓来源的肥大细胞 β-己糖胺酶 类胰蛋白酶 

分 类 号：R392.12[医药卫生—免疫学] R392-33[医药卫生—基础医学]