流式细胞术监测免疫抑制后树鼩外周血淋巴细胞的动态变化  

Dynamic changes of peripheral blood lymphocytes in tree shrews after immunosuppression monitored by flow cytometry

作　　者：江惠 赖永静 李骅 冯一唯 夏巍 唐安洲[1] Jiang Hui;Lai Yongjing;Li Hua;Feng Yiwei;Xia Wei;Tang Anzhou(Department of Otorhinolaryngology Head and Neck Surgery,The First Affiliated Hospital of Guangxi Medical University,Key Laboratory of Early Prevention and Treatment of Regional High Incidence Tumors,Ministry of Education,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学第一附属医院耳鼻咽喉头颈外科,广西医科大学区域性高发肿瘤早期防治研究教育部重点实验室,南宁530021

出　　处：《广西医科大学学报》2022年第6期874-879,共6页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.32060132)。

摘　　要：目的:探索建立一种适用于树鼩外周血淋巴细胞的流式细胞学方法,并利用该方法动态监测注射环孢素A(CsA)树鼩的淋巴细胞、CD4^(+)细胞、CD19^(+)细胞百分比的变化。方法:将18只成年树鼩随机分为空白组(10只)、CsA高剂量组(4只)和CsA低剂量组(4只)。CsA高、低剂量组分别经腹腔注射20 mg/kg和40 mg/kg的CsA,连续注射35 d。分别在第1天和第7天采集空白组树鼩的股静脉血,进行不同时间淋巴细胞及各亚群差异分析,以检验流式实验操作的稳定性。分别用PBS溶液和含1%牛血清白蛋白(BSA)的PBS溶液重悬外周血单细胞,台盼蓝染色法检测两种不同染色缓冲液对细胞存活率的影响。流式细胞术检测树鼩外周血淋巴细胞、CD3^(+)细胞、CD4^(+)细胞和CD19^(+)细胞百分比,观察树鼩在注射低剂量和高剂量CsA后外周血淋巴细胞、CD4^(+)及CD19^(+)细胞的变化趋势。结果:采用含1%BSA的PBS作为染色缓冲液,可使流式细胞存活率保持在90%~100%,高于PBS(P<0.05)。通过不同操作时间的稳定性实验,筛选出适用于树鼩的CD4和CD19抗体;注射CsA后树鼩外周血淋巴细胞、CD4^(+)和CD19^(+)细胞百分率总体呈下降趋势。结论:本研究建立了一种应用流式细胞术检测树鼩外周血淋巴细胞的方法,为研究树鼩疾病模型的免疫学特性提供动态观察方法。Objective:To establish a flow cytometry method for peripheral blood lymphocytes of tree shrew,and to use this method to dynamically monitor the percentage of lymphocytes,CD4^(+) and CD19^(+) cells in tree shrew injected with Cyclosporine A(CsA).Methods:Eighteen adult tree shrews were randomly divided into blank group(n=10),CsA high-dose group(n=4),and CsA low-dose group(n=4).CsA high-dose and low-dose groups were intraperitoneally injected with 20 mg/kg and 40 mg/kg of CsA for consecutive 35 days,respectively.The femoral venous blood of tree shrews in the blank group was collected on the 1st and 7th day,respectively,and the difference of lymphocytes and subgroups at different times were analyzed to test the stability of flow cytometry.Peripheral blood single cells were resuspended in PBS solution and PBS solution containing 1%bovine serum albumin(BSA),respectively,and the effect of two different staining buffers on cell viability were detected by Trypan blue staining.Flow cytometry was used to detect the percentages of lymphocytes,CD3^(+) cells,CD4^(+) cells,and CD19^(+) cells in the peripheral blood of tree shrew,and the changes of peripheral blood lymphocytes,CD4^(+) and CD19^(+) cells were observed after injection of low-dose and high-dose CsA.Results:Using PBS containing 1%BSA as the staining buffer,the survival rate of flow cytometry cells could be maintained at 90%to 100%,which was higher than that of PBS(P<0.05).The CD4 and CD19 antibodies that suitable for tree shrew were screened by stability experiments at different operating times.The percentage of peripheral blood lymphocytes,CD4^(+) and CD19^(+) cells in tree shrew were decreased after CsA injection.Conclusion:This study established a flow cytometry method for detecting peripheral blood lymphocytes of tree shrew,which provided a dynamic observation method for study of immunological characteristics of tree shrew disease models.

关 键 词：树鼩 流式细胞术 淋巴细胞 

分 类 号：R446[医药卫生—诊断学]