红螯光壳螯虾(Cherax quadricarinatus)血细胞生成的初步研究

A preliminary study on hemopoiesis of Cherax quadricarinatus

为了揭示失血与LPS刺激后红螯光壳螯虾血细胞的生成及其成熟过程,利用EdU标记检测失血后红螯光壳螯虾造血组织细胞增殖的变化,并追踪标记新生血细胞产生的释放情况.利用流式细胞技术将红螯光壳螯虾的4种类型血细胞(透明细胞、小颗粒细胞、中颗粒细胞和大颗粒细胞)按大小进一步分为Ⅰ~Ⅳ类,检测分析失血和LPS刺激后螯虾不同类型血细胞的比例变化.结果显示:红螯光壳螯虾造血组织细胞在失血刺激6~24 h时,无论失血与否,造血组织都处于活跃的增殖状态,增殖水平无明显差异.失血后,血淋巴中出现的EdU标记的新生血细胞较其他血细胞小且圆,推测其为前血细胞.透明细胞和小颗粒细胞主要分布在300~400μm2之间,中颗粒细胞和大颗粒细胞主要分布在400~500μm2之间,随着血细胞颗粒性的增加,细胞逐渐增大.失血刺激12 h时,透明细胞和小颗粒细胞显著增加,而中颗粒和大颗粒细胞显著减少.透明细胞和小颗粒细胞的增加主要由各自的Ⅰ类细胞比例显著增加造成,说明失血后红螯光壳螯虾启动造血最先产生释放的为此类较小的前血细胞.LPS刺激36 h时,红螯光壳螯虾小颗粒细胞比例显著升高,大颗粒细胞比例显著降低.进一步分析发现,LPS刺激12 h时,透明细胞和小颗粒细胞中的Ⅱ类细胞比例明显降低,至24 h时Ⅰ类和Ⅱ类细胞的比例开始显著升高.由此认为,失血刺激与LPS刺激可能通过不同方式作用于螯虾血细胞的释放及成熟.

To explore the production and maturation of hemocytes in Cherax quadricarinatus after haemolymph loss and LPS stimulation,EdU labeling was used to detect the proliferation of hemopoietic tissue and trace the production and release of new hemocytes of C. quadricarinatus after haemolymph loss. Then,the four types of hemocytes(hyaline cells,small,medium and large granular cells)were divided into Ⅰ-Ⅳ categories respectively according to their size by flow cytometry to detect and analyze the proportion changes of hemocytes of C. quadricarinatus after haemolymph loss and LPS stimulation. It was found that the hemopoietic tissue of C. quadricarinatus was in an active proliferative state no matter whether the haemolymph was lost or not when it was stimulated by haemolymph loss for 6-24 h. and there was no significant difference in proliferation.The new hemocytes labeled by EdU,which were smaller and rounder than other hemocytes,appeared in the hemolymph of C.quadricarinatus after haemolymph loss. It was speculated that the new hemocytes might be prohemocytes. It was found that the proportion of hyaline cells and small granular cells mainly distributed in the range of 300-400 μm~2,and the medium and large granular cells mainly distributed in the range of 400-500 μm~2. As the granularity of hemocytes increased,the size of hemocytes gradually increased. At 12 h after haemolymph loss,hyaline cells and small granular cells increased significantly,and medium and large granular cells decreased significantly. Further analysis revealed that the increase of hyaline cells and small granular cells was mainly caused by the significant increase in the proportion of type Ⅰ cells,suggesting that these smaller prohemocytes were the first to be produced and released after haemolymph loss. Small granular cells increased significantly,and large granular cells decreased significantly at 36 h after LPS stimulation. Further analysis revealed that the proportion of type Ⅱ cells of hyaline cells and small granular cells decreased significantly at 12 h,and then the proportion of type Ⅰ and Ⅱ cells increased significantly at 24 h. In short,haemolymph loss and LPS stimulation may act on the release and maturation of hemocytes in C. quadricarinatus in different ways.