CFSE标记法分析番鸭呼肠孤病毒感染对番鸭回肠淋巴细胞归巢的影响

旨在建立一种可靠的体外活番鸭淋巴细胞的荧光标记方法，并用于分析番鸭呼肠孤病毒感染对雏番鸭回肠淋巴细胞归巢的影响。选择活细胞荧光染剂5（6）-羧基二乙酸荧光素琥珀酰亚胺酯（CFDA-SE，CFSE）对分离的番鸭淋巴细胞进行标记和利用流式细胞术对体外标记的淋巴细胞体内示踪检测分析，并利用该方法对MDRV感染雏番鸭回肠组织的淋巴细胞数量进行流式细胞术和石蜡切片免疫荧光检测分析；结果最终确定CFSE体外标记番鸭淋巴细胞的条件为以PBS为孵育液，终浓度10 μmol·L^-1，37℃ 30 min；试验结果表明番鸭外周血内的CFSE⁺淋巴细胞率基本稳定在2%~5%，CFSE⁺淋巴细胞峰值出现顺序依次为脾、空肠、回肠、盲肠、十二指肠、法氏囊和胸腺；此外，感染MDRV后1~10 d MDRV组中CFSE⁺淋巴细胞率极显著（P<0.01）高于MOCK组，该结果与α4⁺淋巴细胞率和石蜡切片免疫荧光检测结果一致。结果表明本试验CFSE标记的淋巴细胞可用于体内淋巴细胞示踪，初步应用结果提示MDRV感染促进番鸭淋巴细胞向回肠归巢，为进一步阐明MDRV感染的肠道组织致病机制奠定了基础。

Analysis of the Effects of Muscovy Duck Reovirus on Lymphocyte Homing in Muscovy Ducklings Ileum by CFSE-Labeling Assay

The aim of this study was to establish a simple and reliable assay for fluorescence labeling Muscovy duck lymphoeytes and then analyze the effects of Muscovy duck reovirus (MDRV) infection on lymphocytes homing of ileum in Muscovy ducklings. 5(6)-carboxydiacetate fluorescein succinimidyl ester (CFDA-SE, CFSE), a living cell fluorescent reagent, was used to label the peripheral blood lymphocytes of Muscovy ducklings. Flow cytometry was used to detect and analyze the lymphocytes that marked in vitro and traced in vivo. In addition, CFSE⁺ lymphocytes of ileum in MDRV-infected and uninfected Muscovy duck were detected and analyzed by flow cytometry and paraffin section immunofluorescence. Results were as follows:The optimal conditions of labeling lymphocytes by CFSE were PBS as the incubation solution, 10 μmol·L^-1 CFSE, 37℃ and 30 min, respectively. The labeled lymphocytes were injected into the Muscovy duck through the brachial vein, the CFSE⁺ lymphocytes in the peripheral blood were basically stable at 2% to 5%. The order of sequence of peak of CFSE⁺ lymphocytes was spleen, jejunum, ileum, cecum, duodenum, bursa and thymus. CFSE-labeled lymphocytes were injected into brachial veins of the young Muscovy ducks before MDRV infection, and the rate of CFSE⁺ lymphocytes in MDRV group was significantly (P<0.01) higher than that in the MOCK group at 1-10 days post infection. This result was consistent with tests of α4⁺ lymphocytes and paraffin section fluorescence. Overall, these results showed that the CFSE-labeling lymphocytes can be used for lymphocytes trace in vivo, and preliminary application results revealed that MDRV infection promotes the lymphocyte homing of ileum in Muscovy ducklings, which provide a basis for further studying the intestine pathogenic mechanism of MDRV infection.