摘要
目的建立保健食品增强免疫力功能评价指标的流式细胞术检测方法,并初步评价其应用价值。方法3种受试物蛋白粉、参茸膏和灵芝虫草菌丝体小鼠实验模型的实验剂量组均按人体推荐量的5、10、30倍进行灌胃,阴性及阳性对照组均给予等量纯净水,阳性对照组为给药结束前3d开始每天灌胃给予25mg/kg左旋咪唑,并于给药结束当天用2%绵羊红细胞进行免疫;大鼠实验模型采用拌饲法按人体推荐量的25、50倍给予受试物;实验结束后同步检测以下指标:(1)传统指标:包括刀豆素A诱导的小鼠脾淋巴细胞转化实验、小鼠脾自然杀伤(NK)细胞活性测定、绵羊红细胞诱导的小鼠迟发型变态反应实验、小鼠腹腔巨噬细胞吞噬鸡红细胞实验。(2)流式细胞仪检测指标:小鼠外周血淋巴细胞计数,大、小鼠T、NK细胞表面活化抗原检测以及小鼠腹腔巨噬细胞吞噬荧光微球功能。结果给予受试物蛋白粉后,0.83、1.67、5.01g/kg剂量组小鼠T、B淋巴细胞的相对比例和绝对数量与阴性对照组间差异无统计学意义;给予受试物参茸膏后,3.75、7.50和15.0ml/kg剂量组的小鼠外周血T淋巴细胞CD69表达阳性比例较阴性对照组明显升高,与其他传统检测指标有良好一致性;蛋白粉0.83、1.67g/kg剂量组小鼠外周血NK细胞CD69表达阳性比例较阴性对照组明显升高,且与NK细胞活性测定结果有良好一致性;给予1.50g/kg剂量灵芝虫草菌丝体的大鼠外周血NK细胞CD25表达阳性比例较阴性对照组升高;小鼠腹腔巨噬细胞吞噬功能实验:传统检测方法未发现对照组与剂量组有明显差别,而流式细胞术检测结果显示:灵芝虫草菌丝体0.15、0.30和0.90g/kg剂量组总吞噬比例增加,0.30、0.90g/kg剂量组吞噬指数均提高。结论应用流式细胞术检测保健食品增强免疫力功能指标,与传统方法检测结果具有良好一致性,且具有更高的敏感性,在检测和评价保健食品增强免疫力功能方面具有良好的应用价值,值得进一步研究和推广。
Objective To establish flow cytometry （FCM） methods and evaluate their application value for measuring the index for enhancing immune function of health food. Methods In mice experiment model, the dosage groups were respectively oral fed with three test substances according to 5, 10, 30 times of the recommended dose for humanbody; both the negative and positive control groups were fed with equivalence purified water once a day. The positive control was fed with 25 mg/kg body weight levamisole for 3 days before finishing the administration, and the immuned two percent of sheep erythrocytes were administrated at the last day. In rats experiment model, the test substance was given by mixing feed according to 25 and 50 times of the recommended dose for humanbody. At the end of the experiment, indices below were simultaneously detected. （1） The classical indices included： spleen lymphocyte transformation test by using ConA （MTT assay）; spleen NK cell activity test （LDH assay）; delayed-type hypersensitivity test by using sheep erythrocyte （foot palm thickening） method and phagocytosis activity tested by mice peritoneal macrophages. （2） FCM indices included： T and B lymphocytes quantitating in mice peripheral blood, activated antigen expression level in the surface of T lymphocytes and NK cells and phagocytosis activity for fluospheres in mice peritoneal macrophages. Results （1） Compared with the negative control group, there were no significant differences in T and B lymphocytes proportion and the number of lymphocytes in mice peripheral blood after given 0.83, 1.67, 5.01g/kg protein powder; （2） mice peripheral blood T lymphocyte sub-cluster CD^＋69/CD^＋3 of 3.75, 7.50, 15.0ml/kg bw Cen-Rong Cream groups were all significantly increased （P〈0.05 ）, which were shown a good coherence with the classic test index; （3） mice peripheral blood NK cell sub-cluster CD^＋69/NKG2D^＋ of 0.83, 1.67 g/kg protein powder groups were both significantly increased （P〈0.05 ）, which was kept in good coherence with those of NK cell activity test （LDH assay）; rats peripheral blood NK cell sub-cluster CD161＋/CD^＋25 of 1.50 g/kg ganodermalucidum and cordycepicmycelia group was significantly increased （P〈0.05 ）; （4） the phagocytosis activity in mice peritoneal macrophages： there were no significant difference found between the controls and the dosage groups in the classic test. However, in the FCM test, the percentage of phagocytic cells of 0.15, 0.30, 0.90 g/kg ganodermalucidum and cordycepicmycelia groups and the phagocytic index of 0.30, 0.90g/kg were enhanced. Conclusion It suggests that was shown in detecting and assessing enhancing immune function of health food the results tested by FCM were fairly consistent with those by using traditional methods, most of them would have higher sensitivity. It should be valuable to applying FCM in the measurement and assessment of enhancing immune function of health food and worth while to further study as to enlarging its application.
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2005年第5期335-341,共7页
Chinese Journal of Preventive Medicine
基金
广东省重点医学科技攻关专项课题(粤卫[2003]246号)
广东省医学科学技术研究基金(A2003074)
