小鼠肝脏淋巴细胞几种分离方法的比较  

Comparison of several methods for the isolation of murine hepatic lymphocytes

作　　者：孔晓明[1] 金齐力[1] 韦莉[1] 王婷[1] 范伟[1] 朱翔[1] 刘勇[1] 

机构地区：[1]蚌埠医学院病原生物学教研室安徽省感染与免疫重点实验室,安徽蚌埠233030

出　　处：《蚌埠医学院学报》2013年第8期1052-1055,共4页Journal of Bengbu Medical College

基　　金：安徽省高等学校省级优秀青年人才基金资助项目(2010SQRL124)

摘　　要：目的:通过比较几种肝脏淋巴细胞分离方法得到的总淋巴细胞、T细胞、自然杀伤T细胞(NKT cells)及自然杀伤细胞(NK细胞)的比例和细胞绝对数,明确各种方法的适用范围。方法:按照析因设计,将不放血(A法)、摘眼球放血(B法)、体循环灌流(C法)、摘眼球结合体循环灌流(B法+C法)4种肝脏预处理方法和单浓度离心法(Ⅰ法)、非连续密度梯度离心法(Ⅱ法)2种Percoll分离方法进行全面组合,共获得8种组合条件,分离小鼠肝脏淋巴细胞,用流式细胞术检测各类型细胞比例。结果:肝脏预处理方法中B法+C法获得淋巴细胞比例最高,A法获得淋巴细胞绝对数最高,B法获得的NKT细胞和NK细胞比例和细胞绝对数均最高;Ⅱ法获得的淋巴细胞、T细胞、NKT细胞和NK细胞比例和细胞绝对数均高于Ⅰ法。对于NKT细胞比例和NK细胞绝对数,肝脏预处理方法和Percoll浓度之间有交互作用(P<0.01和P<0.05),其他均无交互作用(P>0.05)。结论:采用摘眼球放血结合体循环灌流法,结合Percoll非连续密度梯度法(33%+70%),可获得较高数量和比例的固有免疫细胞,有助于对肝脏固有免疫细胞如NKT细胞、NK细胞等的研究。Objective： To compare and define the suitable range of the different methods for isolation of murine hepatic lymphocyte,natural killer T cells（NKT cells） and natural killer cells（NK cells）.Methods： According to factorial design,four liver pretreatment methods including unbleeding method（A method）,the eyeballs bleeding method（B method）,body perfusion method（C method）,the eyeball with body perfusion method（B method ＋ C method） and two percoll isolation methods including single concentration method（Ⅰ method） and continuous density gradient method（Ⅱ method） had been combined into 8 kinds of combination conditions,four bleeding methods were used to prepare cell suspension respectively,and the single concentration method（33% Percoll） and the continuous density gradient method（33% Percoll ＋ 70% Percoll） were used to separation of mouse hepatic lymphocytes respectively,the hepatic innate immune cells were analyzed with two or three-colors immunofluorescent-labeling.Results： With the analysis of variance of factorial design,the highest proportion of lymphocytes was obtained by B method ＋ C method,the highest absolute lymphocyte count was obtained by A method,the highest NKT and NK cell percentage and absolute number was obtained through B method;the ratios and the number of lymphocyte,T cell,NKT cell,NK cell obtained from B method were higher than those of A method.The ratio of NKT cell and NK cell absolute count,liver pretreatment method and percoll concentration had interactions each other（P 0.01 and P 0.05）,the others were no interaction（P 0.05）.Conclusions： High proportion of natural immune cells can be obtained by bleeding with perfusion combined with the continuous density gradient method（33% ＋ 70%）,it is helpful for research on NKT cells and NK cells.

关 键 词：细胞分离 肝脏淋巴细胞 分离方法 PERCOLL 析因分析 

分 类 号：R446.113[医药卫生—诊断学]