| 应用流式细胞术检测虾类血细胞总数方法的建立 |
| Measurement of Total Haemocyte Count in Shrimp by Flow Cytometry |
| 投稿时间：2014-11-21 修订日期：2014-12-23 |
| DOI：10.15928/j.1674-3075.2015.03.015 |
| 中文关键词:血细胞总数 流式细胞术 虾类 镉 血细胞 |
| 英文关键词:total haemocyte count flow cytometry shrimp cadmium haemocyte |
| 基金项目:国家自然科学基金(31302164)；广东省自然科学基金(S2012040008093)；中国热带农业科学院热带生物技术研究所基本科研业务费专项资金(ITBB2015ZY01)。 |
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| 中文摘要: |
| 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。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/mL和（15.09±1.76）×106个/mL，2种方法测定结果的相关性极显著。凡纳滨对虾经不同浓度（0.5 mg/L和5 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 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%，表明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。 |
| 英文摘要: |
| The circulation of haemocytes in shrimp (total haemocyte count, THC) is essential to the immune system of shrimp and is readily influenced by environmental factors. The THC is widely used to indicate the condition of the immune system and as an indicator of environmental stress. In this study, we optimized the flow cytometry (FCM）method for quickly and accurately measuring the total haemocyte count in Litopenaeus vannamei. SYBR Green I was used as a probe for viable haemocytes. Three different doses (1×, 10× and 100×) of SYBR Green I were tested and the fraction of dyed haemocytes was determined at different incubation times. Results show that the optimal dose of SYBR Green I and incubation time were 10× and 60 min. To demonstrate its efficacy, the THC of 10 white shrimps was determined by both flow cytometry (FCM) and microscopic examination and the mean values, (16.68±1.57)×106 cells/mL and (15.09±1.76)×106 cells/mL, were highly correlated. Then the optimized method was used to measure the effect of cadmium stress on the THC of Litopenaeus vannamei. The white shrimps were exposed to two doses of Cd2+ (0.5 and 5 mg/L) and the THC decreased with exposure time. After 48 hr, the THC of white shrimps exposed to 0.5 mg/L and 5 mg/L Cd2+ decreased to 78.7% and 64.7% of those in the control groups, indicating that Cd2+ was toxic to shrimp haemocytes and lowered haemocyte activity, consistent with previous studies. The results demonstrate that the optimized method can be used effectively in cytological studies of shrimp haemocytes. |
| 冼健安,张秀霞,郭慧,钱坤,王安利,王冬梅.2015.应用流式细胞术检测虾类血细胞总数方法的建立[J].水生态学杂志,36(3):89-94. |
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