应用流式细胞术检测虾类血细胞总数方法的建立

通过对流式细胞术(FCM)检测虾类血细胞总数(THC)的条件和方法进行优化,为虾类血细胞学研究提供快捷、准确的测定方法。2012年7-9月,应用SYBR Green I作为荧光染料标记完整血细胞,设置3个不同染料浓度(1×、10×和100×),测定不同孵育时间下染色细胞比例的变化。结果显示,染料终浓度为10×时染色效果最佳,其最佳孵育时间为60 min;应用建立的FCM方法和显微计数方法测定10尾凡纳滨对虾(Litopenaeus vannamei)的THC,平均值分别为(16.68±1.57)×106个/m L和(15.09±1.76)×106个/m L,2种方法测定结果的相关性极显著(R2=0.8064,P<0.01)。凡纳滨对虾经不同浓度(0.5 mg/L和5.0 mg/L)的Cd2+胁迫,利用建立的FCM方法测定Cd2+胁迫下对虾THC的变化。结果显示,0.5 mg/L和5.0 mg/L Cd2+胁迫下,对虾THC随着胁迫时间的延长不断下降,胁迫48 h时分别下降至对照组的78.7%和64.7%,可见Cd2+胁迫对虾类血细胞产生毒性,抑制了血细胞活性,表明该方法适用于虾类的血细胞学研究。

Measurement of Total Haemocyte Count in Shrimp by Flow Cytometry

In order to provide a fast and accurate method for cytological study of shrimp haemocyte, a flow cytometric method for measurement of the total haemocyte count (THC) wasestablished. Results showed that the optimal dose and incubation time were 10× and 60 min respectively, when SYBR Green I was used as a probe for live haemocytes. The white shrimp Litopenaeus vannamei were exposed to different doses (0.5 and 5 mg/L) of Cd2+, and then change of THC was analyzed using the previous method. Results showed that Cd2+ at doses of 0.5 and 5 mg/L depressed the THC of shrimp, indicating that Cd2+ is cytotoxic for shrimp haemocytes, which restrain the haemocyte viability. The results also demonstrate that the established method is feasible for cytological study of shrimp haemocyte.