浅议小鼠腹腔巨噬细胞吞噬乳胶颗粒实验条件筛选（Screening of experimental conditions for phagocytosis of latex particles by mouse peritoneal macrophages）

浅议小鼠腹腔巨噬细胞吞噬乳胶颗粒实验条件筛选（Screening of experimental conditions for phagocytosis of latex particles by mouse peritoneal macrophages） 浅议小鼠腹腔巨噬细胞吞噬乳胶颗粒实验条件筛选(Screening of experimental conditions for phagocytosis of latex particles by mouse peritoneal macrophages) Screening of experimental conditions for phagocytosis of latex particles by mouse peritoneal macrophages [Abstract] Objective To establish and improve a reliable, sensitive and stable method for evaluating phagocytosis of peritoneal macrophages. Methods the mouse peritoneal macrophages and latex particles in different culture conditions with 24 quantitative plate, after mixing 37 C, 5% CO2 incubator after incubation, fluorescence microscope to count the percentage and index of phagocytosis. Results with the incubation time (0.5, 1, 2, 4 h) prolonged, phagocytic rate of peritoneal macrophages phagocytosis of latex particles increased between the phagocytic percentage of different culture time differences were statistically significant (P<0.05); with calf serum concentration (2.5%, 5%, 10%) and latex particle concentration (0.25 per thousand, 0.5 per thousand. 1 per thousand, 2 per thousand) increased peritoneal macrophage phagocytic percentage and phagocytic index increased between phagocytic percentage and phagocytic index showed significant differences in different concentrations of calf serum and abdominal cavity macrophage latex particles (P<0.01); peritoneal macrophages adherent after treatment of phagocytic percentage and phagocytic index was significantly higher than that of untreated adherent cells treated with P<0.01 (); at Balb/c\, C57, Kunming (KM), ICR of the 4 strains of mice, Balb/c mice peritoneal macrophage phagocytosis percentage was significantly higher than that of other strains of mice (P <0.01). Conclusion the concentration of latex particles is 1 per thousand, serum concentration is 2.5% to 5%, cell wall adherent 2 h, phagocytosis culture 2 h, and Balb/c mice is the best experimental condition. Keywords latex particles; peritoneal macrophages; phagocytosis index; experimental conditions; Study on experimental conditions for phagocyteosis of mouse peritoneal macrophages WANG Hui, LEI Zhi Ming, HAO Wei and dong.Department of Toxicology, School of Public Health Peking University (Beijing 100191, China) Abstract:Objective To provide a reliable, sensitive and stable method for detecting the percentage of phagocytosis of mouse peritoneal macrophages.Methods The mouse peritoneal macrophages were incubated with latex beads in plate in cell culture 24 well incubator.The percentage of phagocytosis (PP) and phagocytosis index (PI) were detected by fluorescence microscope.Results As the culture time increasing, PP of macrophages became higher (P<0.05).The PP of peritoneal macrophages increased with the increment of calf serum concentration.As the concentration of latex beads increasing, the PP of peritoneal macrophages also increased (P<0.01).The PP of peritoneal macrophages of adherent macrophages was higher than that of non adherent (macrophages P<0.01.The of peritoneal macrophages) PP of the Balb/c mice was significantly higher than that of the C57, KM and ICR mice (P<0.01).Conclusion The optimal experimental conditions of the phagocyteosis of mouse peritoneal macrophages might be the concentration of latex beads of the calf serum concentration 1. Of 2.5%-5%, adding the latex beads and continuous culturing for 2 hours at the time of after the adherence of 2 hours macrophages.Besides, it is indicated that Balb/c mouse is most suitable for detection of the capacity www.seo27.cn of phagocyteosis. Key words:latex beads; 腹腔巨噬细胞;吞噬指数;实验条件 巨噬细胞的吞噬功能是宿主对外源化学物早期关键应答〔1〕。腹腔巨 噬细胞吞噬鸡红细胞试验，是一种广泛应用于检测非特异性免疫功能 的传统方法，主要评价指标为吞噬百分率和吞噬指数〔2〕。由于近年 来对于动物保护的重视和对“3R”原则“还原(减少)”、”置换(替代)”、”细化(优化)”的倡导〔3〕，各国研究人员均在进行替代 试验方法的研究。腹腔巨噬细胞吞噬乳胶颗粒的试验作为一种替代方 法，与传统方法比较可以减少鸡红细胞的使用，并且操作程序简单， 结果判读准确为进一步完善该方法的实验条件，本研究观察不了不同 培养条件和目。前常用的4种品系小鼠对腹腔巨噬细胞吞噬乳胶颗粒 的百分率和吞噬指数的影响，旨在为 标准 excel标准偏差excel标准偏差函数exl标准差函数国标检验抽样标准表免费下载红头文件格式标准下载 化实验方法提供依据。 1材料与方法 1.1材料 1.1.1级雌性实验动物SPF BALB/c小鼠，体重20,22 g，10只、C57小鼠(20,22 g)5只、ICR小鼠体重22,24 g，5只、公里小鼠体 重22,24 g，5只[北京大学医学部实验动物科学部，许可证号:SCXK(京)2006-2008年]。在对不同培养条件对小鼠腹腔巨噬细胞吞噬 功能影响的试验中，除品系对腹腔巨噬细胞吞噬乳胶颗粒百分率的影 响使用4种品系的小鼠外，其余比较均使用BALB/c小鼠。 www.yixueda.com医学论文网 1.1.2主要试剂与仪器乳胶颗粒(美国西格玛公司);RPMI(罗斯威尔公园纪念研究所)1640培养液(美国Gibco公司);小牛血清(北京元亨金马生物技术有限公司)。CO2培养箱(德国但希略斯细胞公司);超净工作台(北京昌平长城空气净化 工程 路基工程安全技术交底工程项目施工成本控制工程量增项单年度零星工程技术标正投影法基本原理 公司)。 1.2方法 1.2.1小鼠腹腔巨噬细胞的制备脱臼处死小鼠，将3毫升小牛血清浓度为5%的RPMI 1640完全培养液注入小鼠腹腔，轻揉腹部。打开腹腔吸取腹腔液，1%冰醋酸计数细胞后用培养液调整细胞浓度为1×106 /毫升。将细胞液放入24孔板中，0.5毫升/孔，37?、5% CO2培养箱中孵育2 h，待细胞贴壁后弃上清，用无血清的冷RPMI 1640冲洗掉未贴壁的细胞〔4〕。 1.2.2条件筛选(1):37培养时间将处理后的细胞在?、5% CO2培养箱分别培养0.5,1,2,4 H后终止试验。(2)小牛血清浓度:加入不同小牛血清浓度的RPMI 1640培养液(2.5%，5%，10%)0.5毫升，乳胶颗粒浓度为1‰，于37?、5% CO2培养箱中培养2 h中止试验。(3)乳胶颗粒浓度:在细胞已贴壁的24孔板中加入含乳胶颗粒浓度分别为0.25‰，0.5‰，1‰，2‰的RPMI1640培养液各0.5毫升，于37?、5% CO2培养箱中培养2 h后中止试验。(4)贴壁与否:不经贴壁处理的细胞直接在含1‰乳胶颗粒的RPMI1640完全培养液中培养2 h后中止实验。贴壁组经贴壁2h后置于含1‰乳胶颗粒的rpmi1620完全培养液中培养2 h后中止试验。(5)品系:不同品系小鼠腹腔巨噬细胞贴壁后，每孔加入含1‰的乳胶颗粒培养液0.5毫升，培养2 h后中止实验。以上除观察不同小牛血清含量对小鼠腹腔巨噬细胞吞噬百分率和指数影响外，其他试验中RPMI 1640培养液的小牛血清含量为采用荧光显微镜下计数细胞吞噬百分率和吞噬指数评价最优条件5%。 (6) count by fluorescence microscope: observe and photograph under fluorescence microscope. Each sample (hole) takes at least 5 different visual fields to ensure that enough cells are counted. The excitation wavelength of fluorescence is 485 nm and the emission wavelength is 530 nm. Percentage of phagocytosis (PP) = (phagocytosis of latex particles per macrophage count / macrophage count) x 100%; phagocytosis index (PI) = (phagocytic total number of latex particles / macrophage count) * 100%. 1.3 statistical analysis using SPSS 13 statistical software for t test. 2 Result 2.1 effects of incubation time on the phagocytic index with the incubation time, peritoneal macrophage phagocytosis percentage increased, respectively (42 + 9.43)% and (63.40 + 4.10)% and (82 + 1.87)% and (89.60 + 1.82)%, each training time phagocytic percentage differences were statistically significant (P<0.05). 2.2 the influence of calf serum concentration on phagocytosis index, with the increase of calf serum concentration, the percentage of phagocytosis and phagocytic index of macrophage increased. 2.5%, 5%, 10% of the calf serum concentration, the percentage of phagocytosis were (77.83 + 4.71)%, (79.17 + 4.31)% and (84 + 3.74%); the phagocytic index was (3.01 + 0.79)%, (3.32 + 0.52)% and (3.79 + 0.60)%. In addition to containing 2.5% and 10% calf serum cultured peritoneal macrophages cultured in comparison with significant differences in phagocytic percentage (P<0.05); the rest of the cultured peritoneal macrophages cultured in the phagocytic percentage or the difference between the phagocytic index were not statistically significant. 2.3 the influence of concentration of latex particles on phagocytosis index (Table 1, Fig. 1), with the increase of latex particle concentration, the percentage of peritoneal macrophage phagocytosis gradually increased. The Balb/c of mouse peritoneal macrophages in latex a concentration of 1 per thousand and 0.5 per thousand and 2% and 1% when the phagocytic percentage of comparison, there were no significant differences in other groups; phagocytic percentage and phagocytic index comparison, the differences were statistically significant (P<0.01 or P<0.05). Table 1 Effect of latex particle concentration on the percentage of peritoneal macrophages in mice: Note: the concentration of latex particles is 0.25 per thousand; a, P<0.05, B, P<0.01; compared with 0.5 per thousand; C, P<0.01; compared with 1 per thousand, D, P<0.01. Apparatus