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1 第 17 卷第 4 期生命科学研究灾燥造援员 7 晕燥援 4 圆园 13 年 8 月蕴蚤枣藻杂糟蚤藻灶糟藻砸藻泽藻葬则糟澡 Aug. 圆园 13 小鼠巨噬细胞吞噬功能测定方法的改进及应用摘余虹 ( 重庆医药高等专科学校药学院, 中国重庆 43) 要 : 对巨噬细胞吞噬鸡红细胞活性的体内检测方法进行改进. 收集小鼠腹腔巨噬细胞, 加入不同浓度甘草多糖尧鸡红细胞于 37 益共同孵育 1h, 在倒置显微镜下观察吞噬状态, 统计吞噬百分率和吞噬指数. 并将改进后的体外吞噬方法用于检测酵母多糖对小鼠腹腔巨噬细胞形态和吞噬功能的影响. 结果表明 : 改进后的体外吞噬法测定的结果与传统的体内吞噬法比较, 两者具有显著的相关性 (n=6, P<.1). 酵母多糖对巨噬细胞刺激 1h 后, 与对照组相比, 其吞噬功能显著性增强 ; 巨噬细胞形态上出现被活化的特征. 应用改进后的方法研究巨噬细胞吞噬鸡红细胞, 不需染色, 在模拟生理条件下进行, 保持了细胞活性, 有简便尧成本低以及准确率高的特点, 适用于普通实验室的科研和教学. 关键词 : 巨噬细胞 ; 鸡红细胞 ; 吞噬 ; 体外中图分类号 : Q331 文献标识码 : A 文章编号 : 17 鄄 7847(213)4 鄄 316 鄄 5 Improvement and Application of Assay for Quantitating Phagocytosis of Mice Peritoneal Macrophages YU Hong (Department of Pharmacy, Chongqing Medical and Pharmaceutical College,Chongqing 43, China) Abstract: The traditional in vivo assay for quantitating phagocytosis of murine peritoneal macrophages was improved. The macrophages were collected from the peritoneal cavity of BALB/C mice. The macrophages treated with glycyrrhiza polysaccharide at different concentrations were incubated with chicken red blood cells (CRBC) at 37 益 with 5% CO 2 inthe air. After 1h, the phagocytic percentages and index were calcu 鄄 lated under inverted microscope. Meanwhile, the improved method was utilized to evaluate the effects of zy 鄄 mosan on the phagocytosis and morphology of macrophages. The results indicated that the phagocytic per 鄄 centages and index by the improved assay were significantly correlated with that of in vivo assay (n=6, P<.1). After 1hof zymosan treatment, the phagocytosis was significantly enhanced by zymosan compared with con 鄄 trol; obvious morphological changes of macrophages were observed, those typical morphological features sug 鄄 gested that macrophages might have been activated. The new improved in vitro assay for quantitating phago 鄄 cytosis without staining was performed under the simulated physiological conditions, which has been keeping the cell activities. It is an easy, fast, cost efficient and reliable method suitable for scientific research and teaching application in aroutine laboratory. Key words: macrophage; CRBC; phagocytosis; in vitro CLC number: Q331 Document code: A Article ID: 17 鄄 7847(213)4 鄄 316 鄄 5 (Life Science Research,213,17(4):316 耀 32) 收稿日期 : 213 鄄 5 鄄 6; 修回日期 : 213 鄄 5 鄄 2 基金项目 : 重庆医药高等专科学校自然科学基金资助项目 (2118) 作者简介 : 余虹 (1982 鄄 ), 女, 广西桂林人, 重庆医药高等专科学校药学院讲师, 硕士, 主要从事药学方面的教学和科研工作,E 鄄 mail: yuhong773@163.com. Received date: 213 鄄 5 鄄 6; Accepted date: 213 鄄 5 鄄 2 Foundation item: Natural Science Foundation of Chongqing Medical and Pharmaceutical College (2118) Biographies: YU Hong (1982 鄄 ), female, Guilin of Guangxi province, lecturer of Chongqing Medical and Pharmaceutical College, mainly re 鄄 searches in pharmacy, E 鄄 mail: yuhong773@163.com.

2 第 4 期余虹 : 小鼠巨噬细胞吞噬功能测定方法的改进及应用 317 The activity of macrophages is important to im 鄄 munologic response [1].Phagocytosis, one of the most important host defense mechanisms against invading microorganisms, is a characteristic of the macro 鄄 phage [2].Reliable analysis of the phagocytic activities of macrophages is of great importance in estimating the immunoreactivity of organisms. Numerous stud 鄄 ies have employed particles to qualitatively assess thephagocyticcharacteristicsofmacrophages invivo. For example, chicken red blood cells (CRBC) were given to mice by peritoneal injection. After aspeci 鄄 fied time interval, the mice were beheaded. Peri 鄄 toneal fluid was extracted from each mouse and transferred onto aslide. After rinsing, fixation, and staining, the macrophages are observed under light optics and the numbers of CRBC associated with them are quantitated [3 耀 5].These methods for quantit 鄄 ating phagocytosis tend to be time 鄄 consuming, labo 鄄 rious and hard to observe the phagocytic process. Another analytical technique used for the quantita 鄄 tion of phagocytosis is flow cytometry. This method appears to be more rapid, accurate, and highly re 鄄 producible but more cost 鄄 consuming [6 耀 1]. The aim of this study was to develop an easy, fast, cost efficient and reliable method suitable for scientific research and teaching application in a routine laboratory. 1 Materials and methods 1.1 Materials BALB/C mice, 1 to 12 weeks old, were pur 鄄 chased from the Chongqing Medical University, Chongqing,China.Glycyrrhizapolysaccharideandzy 鄄 mosan were extracted according to CHENG et al [11,12]. Fetal bovine serum (FBS) and RPMI medium 164 was purchased from Sigma (St. Louis, MO, USA). Giemsa was purchased from Beijing Borunlaite Science &Technology Co., Ltd. 1.2 In vivo assay for quantitating glycyrrhiza polysaccharide induced phagocytosis BALB/C mice were separated into four groups: one control group and three experimental groups. The mice of the experimental groups were given with glycyrrhiza polysaccharide (96, 192 and 288 mg/kg) by oral administration for 6days. One hour after final treatment,.2 ml of CRBC with the con 鄄 centration of 5% (V 颐 V) in normal saline (NS) were given to each mouse by peritoneal injection. Thirty minutes later, the mice were beheaded. 1mL of NS was injected into the abdominal cavity of each mouse. The abdomen of each mouse was kneaded for 3min..3 ml of peritoneal fluid was extracted from each mouse and transferred onto aslide. The slides were incubated for 3 min at 37 益 with 5% CO 2 inthe air. The cells were rinsed with normal saline and fixed with formaldehyde for aminimum of 1h. Thereafter, the slides were rinsed with dis 鄄 tilled water, air dried, and stained with Giemsa. A minimum of 2 macrophages in random, sequential fields were observed under inverted microscope and the percentage of macrophages which had phagocy 鄄 tized CRBC was calculated [13]. 1.3 Collection and purification of macrophages BALB/C mice were beheaded. The macrop 鄄 hages were collected from the peritoneal cavity of BALB/C mice by lavaging with 1 ml of RPMI medium 164 (containing 1 %FBS and 5 mg/l of gentamycin sulfate, abbreviated as RPMI 鄄 164). Following centrifugation of the retrieved lavage flu 鄄 ids at 3 伊 gfor 1 min, the cells were resuspended in RPMI 鄄 164 to a concentration of 1 伊 1 6 /ml. Then, the suspension of macrophages was incubated for 2hat 37 益 with 5% CO 2 inthe air. The cells that did not adhere to the surface of the wells were washed out with NS twice. Thereafter, the macrophages were incubated under above condi 鄄 tions [14]. 1.4 Establishment of improved in vitro phago 鄄 cytosis assay A 1 ml suspension of macrophages with the density at 1 伊 1 6 /ml was transferred into the culture flask and added into 3 滋 Lof CRBC with the con 鄄 centration of 5% (V 颐 V) in NS. And then, the gly 鄄 cyrrhiza polysaccharide with different concentration (2, 4, 6 mg/l) was added into the culture flask as tested group and RPMI 鄄 164 was also added into culture flask as control group. Macrophages were incubated with CRBC for 1hat

3 318 生命科学研究圆园 13 年 37 益 with 5% CO 2 in the air. Thereafter, the phagocytic percentages and phagocytic index were calculated under inverted microscope. Every treat 鄄 ment was repeated six times. 1.5 Detection of zymosan induced phagocytosis and cells morphology with the improved method The tested concentration of zymosan in this assay was 5, 1 and 15 mg/l, respectively. The phagocytic activity was measured according to the method described in section 1.4. And the changes of morphological features of cells were ob 鄄 served under inverted microscope. 1.6 Statistical analysis Each experiment was performed six times. Da 鄄 ta are expressed as the mean 依 standard deviation. The statistical analyses of the data utilized SAS 7 In vivo In vitro version 9.1 (SAS, Cary, NC, USA). Levene s test was employed as the homogeneity test of variance. ANOVA followed by Tukey post hoc test was em 鄄 ployed for multiple comparisons between all groups. P<.5 was considered as statistically significant. 2 Results 2.1 Effects of glycyrrhiza polysaccharide on the phagocytosis of macrophages invivo and invitro Fig.1 showed that the in vivo and vitro phago 鄄 cytosis of macrophages, manifested by parameters of phagocytic percentage and index, were all signifi 鄄 cantly improved by glycyrrhiza polysaccharide compared with controls. Furthermore, at a fixed concentration, the in vitro assay data was signifi 鄄 cantly correlated with that of in vivo assay (Table 1). 8 In vivo In vitro Fig.1 Effect of glycyrrhiza polysaccharide on phagocytosis of macrophages Phagocytic percentage; Phagocytic percentage and index. P<.5 compared with control in vivo; P<.5 compared with control in vitro. Table 1 Correlation of phagocytosis assayed with two methods at different concentrations Groups Low dose group Middle dose group High dose group 2.2 Effects of zymosan on the phagocytosis of macrophages in vitro Correlation coefficient of phagocytic percentage (r 1) Notes: r = ; P<.1. Correlation coefficient of phagocytic index (r 2) After 1hof zymosan treatment in vitro, great morphological differences between macrophages and CRBC were observed under inverted microscope (Fig.2). Fig.2A showed that the membrane of macrophage invaginated. Some CRBC were adher 鄄 ing to the surfaces of macrophage and others were being ingested. The macrophage phagocytosing two CRBC could be clearly observed from Fig.2B. As shown in Fig.2C, the macrophage has engulfed many CRBC, and some of CRBC with fuzzy cellular outlines have been digested. Within the tested concentrations, zymosan had the ability to enhance the phagocytosis of mac 鄄 rophages, which had statistical significance com 鄄 pared with control (Fig.3). Our results were consis 鄄 tent with that of XU et al [15]. 2.3 Effect of zymosan on the morphology of macrophages The culture medium 鄄 treated macrophages were nearly round in shape after incubation for 1h(Fig. 4A); in contrast, zymosan 鄄 treated macrophages were

第 4 期余虹 : 小鼠巨噬细胞吞噬功能测定方法的改进及应用 319 7 6 5 4 3 2 1 (C) Fig.

7 6 5 4 3 2 1 5 1 15 5 1 15 mg/l mg/l Fig.3 Effect of zymosan on phagocytosis of macrophages Phagocytic percentage; Phagocytic percentage and index. P<.5 compared with control.

4 第 4 期余虹 : 小鼠巨噬细胞吞噬功能测定方法的改进及应用 (C) Fig.2 The morphology of macrophages cultured with CRBC CRBC adhering to the surfaces of macrophage; Macrophage phagocytosing two CRBC; (C) Macrophage digested several CRBC mg/l mg/l Fig.3 Effect of zymosan on phagocytosis of macrophages Phagocytic percentage; Phagocytic percentage and index. P<.5 compared with control. deformed: cell size obviously increased, cellular boundaries became vague and irregular, and abun 鄄 dant pseudopodia appear (Fig.4B). These typical mo 鄄 rphological features suggested that macrophages might have been activated, which was consistent with aprevious report [16]. Fig.4 The morphological changes of macrophage treated for 1h Medium 鄄 treated; Zymosan 鄄 treated. 3 Discussion Analysis of the functional activity of phago 鄄 cytes is of value in estimating the immunoreactivity of organisms. Here, the traditional in vivo and im 鄄 proved in vitro assays were employed to assess the effect of glycyrrhiza polysaccharide on the phagocy 鄄 tosis of macrophages in our study. As is shown by the data in Fig.1, the phagocytic percentage and in 鄄 dex of these two assays were all significantly in 鄄 creased compared with controls. The results ob 鄄 tained with two analytical methods are consistent. According to our results, the in vitro assay data ob 鄄 tained at the various tested concentrations are high 鄄 er than that of in vivo assay. There are two possible reasons for the differences. Firstly, the tested con 鄄 centrations of in vitro assay, calculated on the as 鄄 sumption of body fluid equaling to 7% of body weight, are equivalent to the maximum concentra 鄄 tions of glycyrrhiza polysaccharide in mice after o 鄄 ral administration with concentrations of 96, 192 and 288 mg/kg, respectively. Due to the absorption and distribution, the concentration of drug in peri 鄄 toneal cavity of mouse after oral administration is reduced. While, the drugs directly interact with macrophages in vitro. Secondly, compared with in vivo assay, the macrophages and CRBC would more fully interact with each other, since they are re 鄄

5 32 生命科学研究圆园 13 年 mained in suspension during the in vitro assay. So, the in vitro assay would better evaluate the func 鄄 tional status of macrophage population than in vivo assay to some degree [17].Our study showed that there were significantly correlations between the results of traditional in vivo and improved in vitro assays. In order to evaluate the utility of the improved in vitro assay, we examined the phagocytosis and morphology of macrophages by zymosan. Our results were consistent with the previous reports [15, 16].These evidences above suggested that the improved in vitro assay is reliable and effective for quantitating phagocytosis. The advantage of this method might be sum 鄄 marized as follows. Firstly, the improved in vitro as 鄄 say for quantitating phagocytosis is cost efficient. The macrophages of one mouse could serve as sev 鄄 eral samples to be tested. Secondly, the experimen 鄄 tal variation of in vitro assay is lower according to our research, which suggests that the improved as 鄄 say is less affected by individual difference. Thirdly, after incubation, the morphological changes of macrophages and CRBC became obvious. The phagocytic status of the macrophage could be ob 鄄 served clearly without staining. Thus, the in vitro assay iseasier to perform and time efficient.fourthly, the experiment without staining was performed un 鄄 der the simulated physiological conditions, which has been keeping the cell activities. Hence, macrophage engulfing CRBC could be dynamically observed and even videoed, which will helps to en 鄄 hance the students understanding of phagocytic process. This method is much suitable for teaching application in aroutine laboratory. Therefore, the new improved in vitro assay for quantitating phagocytosis is far superior in terms of no staining, it is much less expensive, but much more reproducible and experimentally satisfactory. References: [1] 李杰, 蒋昶宇, 项荣. 免疫应答乙型肝炎病毒治疗中的双刃剑 [J]. 生命科学研究 (LI Jie, JIANG Chang 鄄 yu, XIANG Rong. Immune response the double 鄄 edged sword of the treat 鄄 ment of Hepatitis Bvirus infection[j]. Life Science Research), 211, 15(6): 556 鄄 559. [2] ROBYN LR, ROBERT JG. An improved spectrofluorometric assay for quantitating yeast phagocytosis in cultures of murine peritoneal macrophages[j]. Journal of Immunological Methods, 1989, 123(6): 259 鄄 267. [3] 黄泰康援常用中药成分与药理手册 [M]. 北京 : 中国医药出版社 (HUANG Tai 鄄 kang. Handbook of the Ingredient and Phar 鄄 macology of Traditional Chinese Medicine[M]. Beijing: China Medicine Science and Technology Press), 鄄 948. [4] 陈奇. 中药药理研究方法 [M]. 北京 : 人民卫生出版社 (CHEN Qi. Methodology on Pharmacology of Traditional Chinese Me 鄄 dicine[m]. Beijing: People s Medical Publishing House), 鄄 117, 936 鄄 938. [5] 白毓谦. 微生物实验技术 [M]. 济南 : 山东大学出版社 (BAI Yu 鄄 qian. Microbiology Experiment Technology[M]. Jinan: Shandong University Press), 鄄 483. [6] STRINGER B, IMRICH A, KOBZIK L. Lung maerophage up 鄄 take of environmental particulates: aflow cytometric assay[j]. Cytometry, 1995, 2(1): 23 鄄 32. [7] ICHINOSE M, HARA N, SAWADA M, et al. Aflow cytomet 鄄 ric assay reveals an enhancement of phagocytosis by platelet activating factor in murine peritoneal macrophages[j]. Cellular Immunology, 1994, 156(2): 58 鄄 518. [8] DREVETS DA, ELLIOTT AM. Fluorescence labeling of bac 鄄 teria for studies of intracellular pathogenesis[j]. Journal of Im 鄄 munological Methods, 1995, 187(1): 69 鄄 79. [9] BROUSSEAU P, PELLERIN J, MORIN Y, et al. Flow cytome 鄄 try as atool to monitor the disturbance of phagocytosis in the clam mya arenaria hemocytes following in vitro exposure to heavymetals[j]. Toxicology, 2, 142(2): 145 鄄 156. [1] FURUKAWA S, SAITO H, INOUE T, et al. Supplemental glu 鄄 tamine augments phagocytosis and reactive oxygen intermedi 鄄 ate production by neutrophils and monocytes from postopera 鄄 tive patients in vitro[j]. Nutrition, 2, 16(5): 323 鄄 329. [11] 程安玮, 金征宇, 万发春. 甘草多糖对小鼠腹腔巨噬细胞 NO inos 及 inos mrna 表达的影响 [J]. 食品科学 (CHENG An 鄄 wei, JIN Zheng 鄄 yu, WAN Fa 鄄 chun. Effect of glycyrrhiza polysaccharide on expression of inos mrna and generation of NO and inos in mouse peritoneal macrophages[j]. Food Sci 鄄 ence), 29, 3(21): 351 鄄 354. [12] 朱延军, 刘洪梅, 边洪荣, 等. 酵母多糖对小鼠免疫功能影响的体外实验研究 [J]. 安徽农业科学 (ZHU Yan 鄄 jun, LIU Hong 鄄 mei, BIAN Hong 鄄 rong, et al. Study on the effect of yeast polysaccharides on immune function of mice in vitro[j]. Journal of Anhui Agricultural Sciences), 29, 37(28): 鄄 [13] 王根维, 唐仕川. 冬虫夏草水提物对巨噬细胞吞噬功能的实验研究 [J]. 山西医科大学学报 (WANG Geng 鄄 wei, TANG Shi 鄄 chuan. Effects of cordyceps sinensis extracts on macrophage phagocytic function in mice[j]. Journal of Shanxi Medical Uni 鄄 versity), 2, 31(1): 2 鄄 21. [14] WANG DY, YU QH, EIKSTADT P, et al. Studies on adju 鄄 vanticity of sodium houttuyfonate and its mechanism[j]. Interna 鄄 tional Immunopharmacology, 22, 32(2): 1411 鄄 [15] 徐军发, 黄迪南, 吕世静, 等. 酵母多糖对体外培养小鼠腹腔巨噬细胞免疫功能的增强作用 [J]. 广东医学院学报 (XU Jun 鄄 fa, HUANG Di 鄄 nan, L 譈 Shi 鄄 jing, et al. Zymosan enhances the immune function of mouse peritoneal macrophages in vitro[j]. Journal of Guangdong Medical College), 23, 21(3): 199 鄄 2. [16] 荣岳光, 佟建明. 多糖对巨噬细胞功能影响的研究进展 [J]. 中国畜牧兽医 (RONG Yue 鄄 guang, TONG Jian 鄄 ming. Progress of research on effects of polysaccharides on macrophage[j]. China Animal Husbandry &Veterinary Medicine), 27, 34 (5): 9 鄄 13. [17] 汤城, 薛雅蓉, 余飞, 等. 一种检测巨噬细胞吞噬鸡红细胞活性新方法的建立 [J]. 畜牧与兽医 (TANG Cheng, XUE Ya 鄄 rong, YU Fei, et al. Establishment a new method to detect chicken red blood cells phagocytosis of macrophages[j]. Animal Husbandry 驭 Veterinary Medicine), 25, 37(11): 47 鄄 49.

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