【摘要】 摸索改进外用红色诺卡氏菌(Nocardia rubra)细胞壁骨架的两种效力测试方法,即流式细胞术-荧光微球法、流式细胞术-免疫双标法(荧光微球+荧光抗体),并与国家食品药品监督管理局原审批方法进行比较,经过统计学评估,评价两种方法的可行性。通过抽取小鼠免疫后获得的腹腔液注入到流式细胞仪,在发射光的荧光通路中,检测巨噬细胞的荧光强度,测定未吞噬荧光微球的巨噬细胞和吞噬荧光微球的巨噬细胞的比例,全部数据经统计学分析,按公式计算吞噬率及吞噬指数完成流式细胞术检测过程。分别采用荧光微球以及荧光微球+荧光抗体两种方式上机检测巨噬细胞吞噬结果。测得的结果与原检验方法——人工显微镜观察法的结果进行比较,确定评估方法的有效性和准确性。结果表明,显微镜人工计数法与流式细胞术-免疫双标法(荧光微球+荧光抗体)的3次实验结果与正常对照组比较,吞噬率明显升高(P<0.01),吞噬指数明显增大(P<0.01),有显著性差异,判定结果为阳性;3次流式细胞术-荧光微球法实验,结果不稳定。流式细胞术-免疫双标法(荧光微球+荧光抗体)由于加入了成熟小鼠巨噬细胞表面特异性标志物抗体F480荧光抗体,可有效标记小鼠巨噬细胞,加强了流式细胞仪收集巨噬细胞的准确性,两者结合应用更能准确反映供试品对小鼠巨噬细胞吞噬功能的作用。利用此方法检测外用红色诺卡氏菌细胞壁骨架增强免疫力的结果和药品原检验方法的统计学结果一致。该方法获取的巨噬细胞数量远多于原方法,加入的荧光微球及荧光抗体,能更准确反应供试品对小鼠巨噬细胞的吞噬作用。该方法灵敏性高、稳定性好,并且易操作,可作为原实验方法的改良参考。更多还原

【Abstract】 Two kinds of potency testing methods of cell wall skeleton of Nocardia rubra for external use were tried to improve, namely flow cytometry fluorescence microsphere method and flow cytometry double labeling(fluorescence microsphere + fluorescent antibody) method, and compare them with the original approval method of state food and drug administration. After statistical evaluation, the feasibility of the two methods was evaluated. Flow cytometry detection process: the peritoneal fluid obtained after immunization of mice was extracted and injected into the flow cytometry. The fluorescence intensity of macrophages was detected in the fluorescence pathway of emission light, and the proportion of macrophages that did not phagocytize fluorescent microspheres and macrophages that phagocytized fluorescent microspheres were measured. All data were statistically analyzed, and phagocytic rate and phagocytic index were calculated according to the formula. In this method, fluorescent microspheres and fluorescent microspheres + fluorescent antibody were used to detect the phagocytosis of macrophages. The effectiveness and accuracy of the method were evaluated by comparing the results of the original test method with that of the artificial microscope observation method. Compared with the normal control group, the phagocytic rate and phagocytic index were significantly increased(P<0.01) and significantly different(P<0.01) by manual counting under microscope and flow cytometry immunodouble labeling(fluorescent microsphere + fluorescent antibody). The results of three experiments by flow cytometry fluorescent microsphere were not stable. Flow cytometry immunodouble labeling method(fluorescent microsphere + fluorescent antibody) can effectively label mouse macrophages by adding F480 fluorescent antibody, which was a specific marker of mature mouse macrophages, and enhance the accuracy of collecting macrophages by flow cytometry. The combination of the two methods can more accurately reflect the effect of the test substance on the phagocytosis of mouse macrophages. The results of using this method to detect the cell wall skeleton of N. rubra for external use to enhance immunity were consistent with the statistical results of the original drug test method. The number of macrophages obtained by this method was much more than that of the original method. The addition of fluorescent microspheres and fluorescent antibodies can more accurately reflect the phagocytosis of the test sample on mouse macrophages. This method was highly sensitive, stable and easy to operate, which can be used as a reference for the improvement of the original experimental method.更多还原