摘要
为了更精确地对甲壳动物血细胞进行快速分类和功能分析,本研究以中华绒螯蟹为例,依据其血细胞内具有颗粒结构的特征,与常规显微观察对比,探讨了一种基于图像流式细胞仪的血细胞自动化分类新方法,并测量了体质量为(10±3) g的中华绒螯蟹的血细胞对直径1μm微球的吞噬情况。结果显示,两种方法都可将中华绒螯蟹血细胞分为4个类群。显微镜观察分类基于胞内可见颗粒,但由于缺乏精细的量化标准,批次样品的人工辨识结果波动较大;而图像流式方法以胞内全部颗粒结构为对象,利用高精度检测模块测量,结果更准确、客观,而且通量高、重复性好。测量结果显示,中华绒螯蟹血淋巴中无颗粒、小颗粒、中颗粒及大颗粒细胞占比分别为40.62%±2.65%、36.68%±6.84%、7.80%±1.16%和16.51%±5.60%,依据测量的颗粒特征区分的4个类群界限清晰,缺乏过渡样点,提示各类细胞之间可能没有相互转化。进一步的活体微球吞噬实验证实,中华绒螯蟹的4类血细胞都具有吞噬功能,并以无颗粒细胞为主要吞噬类群;吞噬微球的细胞比例在注射后6 h内呈钟型曲线变化,4 h可达峰值(5.69%±0.44%),表明中华绒螯蟹血细胞能高效清除血淋巴中的异物。研究表明,图像流式细胞仪适合于中华绒螯蟹的血细胞分类分析和功能研究,本研究结果为同类研究提供了重要参考,将有助于更全面了解甲壳动物血细胞的功能。
In order to make more accurate classification of crustacean blood cells and understand their phagocytic function more clearly,an imaging flow cytometry-based automatic method for Eriocheir sinensis blood cell classification was established,and this new method was further compared with microscopic observation for evaluating their abilities in classifying blood cells through characteristic differences of cytoplasmic granular structure in cells.On this basis,7.5×10^8 of Thermo Fisher carboxylate-modified yellow-green fluorescent microspheres with 1μm diameter were injected through the heart of E.sinensis of(10±3)g weight;proportions of cells containing engulfed microspheres in hemolymph were measured by imaging flow cytometry at 2,4,6 and 24 h after injection to analyze the phagocytic efficiency and foreign-body-removing ability of blood cells.The results showed that both microscopic observation and imaging flow cytometry-based classification methods could distinguish 4 blood cell populations from hemolymph of E.sinensis,but the population proportions from both methods were different.The possible reasons might be that populations in microscopic observing classification were divided based on the observable granular structures in each cell,but populations got from imaging flow cytometry method were distinguished by all measurable granular structures in each cell.However,due to lack of quantified specific classification standard,the identification results from microscopic observing were fluctuant and what’s more,this work was also time-consuming and laborious compared with automatic methods.On the contrary,imaging flow cytometry measured all detectable intracellular particulate matters.With updated detection module,redesigned flowing system,and,what’s more,innovative integration of photographic function for each detecting channel,imaging flow cytometry could offer a high-throughput detection,and the data were more objective and reliable than those of manual distinguishing and counting.According to the flow cytom
作者
胡锦丽
任星潮
刘逸尘
孙金生
耿绪云
张亦陈
HU Jinli;REN Xingchao;LIU Yichen;SUN Jinsheng;GENG Xuyun;ZHANG Yichen(Tianjin Key Laboratory of Animal and Plant Resistance,College of Life Science,Tianjin Normal University,Tianjin 300387,China;Tianjin Aquatic Animal Infectious Disease Control and Prevention Center,Tianjin 300221,China)
出处
《水产学报》
CAS
CSCD
北大核心
2019年第3期563-572,共10页
Journal of Fisheries of China
基金
国家自然科学基金(31472299)
天津市应用基础与前沿技术研究计划(15JCZDJC33800)
天津市水产生态及养殖重点实验室开放基金(TJAE2015005)
天津市人才发展特殊支持计划高层次创新创业团队项目(ITTFRS2017007)
天津市高等学校创新团队建设规划(TD13-5076)~~
关键词
中华绒螯蟹
血细胞
分类
吞噬
图像流式细胞仪
Eriocheir sinensis
blood cell
classification
phagocyte
imaging flow cytometry
