不同腹腔灌洗液分离培养原代大鼠腹腔巨噬细胞的效率比较及细胞鉴定  

Efficiency Comparison and Cell Identification of Primary Peritoneal Macrophages Isolated from Rats with Different peritoneal Lavage Fluid

作　　者：董晓 柯尊平[3] 周明[1] 王俊峰 党书毅 李军 谭利 DONG Xiao;KE Zun-ping;ZHOU Ming;WANG Jun-feng;DANG Shu-yi;LI Jun;TAN Li(Department of Cardiology, Taihe Hosipital;Key Laboratory of Embryonic Stem Cells;Experimental Teaching Center of Computer, Hubei University of Medicine, Shiyan 442000, Hubei, China)

机构地区：[1]十堰市太和医院.湖北医药学院附属医院心内科 [2]湖北医药学院胚胎干细胞研究湖北省重点实验室 [3]湖北医药学院计算机实验教学中心,湖北十堰442000

出　　处：《湖北医药学院学报》2017年第5期418-421,428,F0003,共6页Journal of Hubei University of Medicine

基　　金：湖北省教育厅指导项目(B2016116);湖北省教育厅科学技术研究项目(Q20132104);湖北省自然科学基金青年项目(2015CFB211);湖北医药学院附属太和医院国家级项目培育基金(2013PY01)

摘　　要：目的:探索最优化的腹腔巨噬细胞分离与培养方法,为体外研究巨噬细胞提供一种简便易行、高效的方法。方法:分别用0.9%氯化钠溶液(对照组)、PBS、无血清高糖DMEM、10%血清高糖DMEM灌洗大鼠腹腔5 min,无菌状态下吸出灌洗液,离心后收集腹腔巨噬细胞,对比不同灌洗液获取细胞数量和活性的差异,通过形态观察、HE染色、吉姆萨染色和CD68免疫组织化学染色对培养的腹腔巨噬细胞进行鉴定。结果:统计每毫升不同腹腔灌洗液获得的细胞数,结果发现PBS组和10%FBS高糖DMEM组与对照组相比差异无统计学意义(P>0.05),无血清高糖DMEM进行腹腔灌洗后获得的细胞数明显多于其他各组(4.13±0.15)×106/m L,差异有统计学意义(P<0.05)。台盼兰染色显示DMEM组[(97.83±0.65)%]和10%FBS DMEM组[(97.90±1.10)%]细胞活性较高,与对照组[(95.50±0.76)%]相比差异有统计学意义(P<0.05),PBS组[(96.17±1.04)%]细胞活性与对照组相比差异无统计学意义(P>0.05),所有灌洗液获得巨噬细胞活性均在95%以上。分离培养的细胞经HE染色、吉姆萨染色和CD68免疫组化染色鉴定为巨噬细胞。结论:无血清高糖DMEM是高效获取大鼠腹腔巨噬细胞的灌洗液,该方法是一种简易实用的体外分离大鼠腹腔巨噬细胞的方法。Objective To explore an optimal method for isolation and culture of peritoneal macrophages,and to provide a simple and efficient method for the study of macrophage in vitro. Methods Rat peritoneal lavage was performed with 0.9%sodium chloride solution（ control group）,PBS,high glucose DMEM and high glucose DMEM contain 10% fetal bovine serum for 5 min at aseptic condition. Then,peritoneal macrophges were collected from lavage aspiration after centrifugation.The number and viability of the cultured peritoneal macrophages from different lavage fluid were identified through morphological observation,HE staining,Giemsa ＇s staining and CD68 immunohistochemical staining. Results Based on the cell counts per milliliter of peritoneal lavage fluid,the results of PBS and high glucose DMEM with 10% FBS groups were not statistically different from that of control group（ P〉0.05）. Compared with other lavage fluid,high glucose DMEM was the most efficient lavage fluid for peritoneal cell collection [（ 4. 13 ± 0. 15） × 106/m L,P 〈 0. 05]. Trypan blue staining showed that the cell viability of peritoneal macrophages collected by high glucose DMEM [（ 97. 83 ± 0. 65） %] and high glucose DMEM contain 10% fetal bovine serum[（ 97. 90 ± 1. 10） %] was higher than that of control group[（ 95. 50 ± 0. 76） %,P〈 0. 05] and there was no statistical difference between control and PBS [（ 96.17±1.04） %]groups（ P〉0.05）. The cell viability of macrophages obtained by all the lavage fluid was above 95%. The isolated cells were identified as macrophage by HE staining,Giemsa＇s staining and CD 68 immunohistochemistry. Conclusion High glucose DMEM was the most efficient lavage fluid for rat peritoneal macrophage collection. We established a simple and practical method to isolate rat peritoneal macrophage.

关 键 词：腹腔巨噬细胞 大鼠 原代培养 鉴定 免疫组织化学 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]