活体小鼠血管内外淋巴细胞快速识别的方法及应用

Method and application of rapid recognition of intravascular and extravascular lymphocytes in living mice

目的探讨建立一种快速识别活体小鼠血管内外淋巴细胞的方法。方法C57BL/6J小鼠随机分为CD45-APCcy7抗体注射组(n=6)、CD45-BV570抗体注射组(n=3)和对照组(n=3)。抗体注射组小鼠经内眦静脉丛注射抗小鼠CD45抗体(3μg/只或5μg/只),对照组小鼠注射同等体积的磷酸缓冲盐液(phosphate buffer saline,PBS),注射后2.5 min摘眼球取外周血,3 min时断颈处死小鼠,取淋巴结(lymph node,LN)、脾脏(spleen,SP)、骨髓(bone marrow,BM)、肝脏和肺脏与外周血一同制备单个核细胞悬液,每组细胞均进行CD4和CD8分子表面染色,流式细胞仪上机检测。结果流式细胞仪检测结果显示CD45-APCcy7抗体注射组小鼠给予3μg/只和5μg/只两个剂量后,外周血中CD4^(+)和CD8^(+)细胞95%以上均为CD45^(+)细胞[CD4^(+)细胞:3μg/只和5μg/只CD45^(+)细胞百分率为(96.20±1.50)%比(98.77±1.88)%,t=1.85,P>0.05;CD8^(+)细胞:3μg/只和5μg/只CD45^(+)细胞百分率为(99.27±0.75)%比(99.93±0.12)%,t=1.52,P>0.05],表明此两种剂量均可实现血管内淋巴细胞几乎全部活体、快速染色。在3μg/只注射剂量下,使用同一克隆号不同荧光标记的抗CD45抗体检测时,外周血中95%以上CD4^(+)和CD8^(+)细胞均可被染色为CD45^(+)。在CD45-APCcy7抗体注射组和CD45-BV570抗体注射组CD4^(+)细胞中CD45^(+)细胞百分率分别为(96.20±1.50)%和(99.43±0.98)%;而CD8^(+)细胞中CD45^(+)细胞百分率分别为(99.27±0.75)%和(99.90±0.17)%均高于对照组(t值分别为111.10,175.40,229.00,994.90,P值均<0.05)。淋巴器官和非淋巴器官染色结果表明,抗CD45抗体可清楚地区分CD45^(+)和CD45-细胞群,两种不同标记的抗体对CD45^(+)细胞染色的百分率分别为LN[(0.35±0.23)%比(0.61±0.13)%;t=1.69,P>0.05]、SP[(1.07±0.28)%比(2.87±1.13)%;t=2.85,P>0.05]、BM[(0.85±0.24)%比(1.68±0.73)%;t=1.89,P>0.05]、肝脏[(15.53±5.20)%比(19.60±9.36)%;t=0.66,P>0.05]和肺脏[(29.97±6.14)%比(41.03±21.78)%;t=0.85,P>0.05],各组织中统计学分析结果差异均无统计学意义。结论本实验建立了一种快速检测并区分血管内外淋巴细胞的方法,且可联合多种标记抗体一起检测,是一种高效、快速、准确的活体小鼠淋巴细胞检测手段。

Objective To establish a method for rapid identification of lymphocytes inside and outside blood vessels of living mice.Methods C57BL/6J mice were randomly divided into CD45-APCcy7 Ab iv group(n=6),CD45-BV570 Ab iv group(n=3)and control group(n=3).Ab iv groups were injected with anti mCD45 Ab(3μg/mouse or 5μg/mouse)and the mice in control group were injected with the same volume of phosphate buffer saline(PBS).After injection for 2.5 min,the peripheral blood from inner canthus vein plexus were collected,and the mice were sacrificed 3 minutes later.The lymph node(LN),spleen(SP),bone marrow(BM),liver and lung were harvested for single nuclear cell suspension;CD4 and CD8 molecules were stained in each group and cells were detected by flow cytometry.Results Flow cytometry results showed that the mice in CD45-APCcy7 Ab iv group were injected with 3μg/mouse or 5μg/mouse,more than 95%of CD4^(+)and CD8^(+)cells in blood were CD45^(+)cells[CD4^(+)cell:CD45^(+)cell percentages of 3μg/mouse and 5μg/mouse are(96.20±1.50)%and(98.77±1.88)%respectively,t=1.85,P>0.05;CD8^(+)cell:CD45^(+)cell percentages of 3μg/mouse and 5μg/mouse are(99.27±0.75)%and(99.93±0.12)%respectively,t=1.52,P>0.05],indicating that almost all of intravascular lymphocytes can be stained quickly in vivo.After 3μg/mouse injection dose with the same clone number of anti-CD45 antibodies labeled with different fluorescence,more than 95%of CD4^(+)and CD8^(+)cells in blood can be stained as CD45^(+).The CD45^(+)cell percentages in CD4^(+)cell of CD45-APCcy7 Ab iv group and CD45-BV570 Ab iv group are(96.20±1.50)%and(99.43±0.98)%respectively;while the CD45^(+)cell percentages in CD8^(+)cell in the two groups are(99.27±0.75)%and(99.90±0.17)%respectively,which were higher than the control group(t values are 111.10,175.40,229.00,994.90,all P values<0.05).The results of lymphatic organs and non-lymphatic organs showed that CD45^(+)and CD45-cell groups can be clearly distinguished by anti-CD45 antibody.The percentages of CD45^(+)cells stained by different labeled anti-CD45 antibodies were LN[(0.35±0.23)%vs(0.61±0.13)%;t=1.69,P>0.05],SP[(1.07±0.28)%vs(2.87±1.13)%;t=2.85,P>0.05],BM[(0.85±0.24)%vs(1.68±0.73)%;t=1.89,P>0.05],liver[(15.53±5.20)%vs(19.60±9.36)%;t=0.66,P>0.05]and lung[(29.97±6.14)%vs(19.60±9.36)%;t=0.85,P>0.05]respectively,and there was no statistical difference in each tissues.Conclusion The experiment establishes a method for rapid detection and identification of lymphocytes inside and outside of blood vessels,and can be combined with a variety of labeled antibodies for further detection,which is an efficient,fast,and accurate method for detecting lymphocytes in living mice.