文蛤(Meretrix meretrix)幼虫发育过程中细胞凋亡和Caspase 功能分析

以文蛤为研究对象,利用TUNEL技术(Terminal-deoxynucleotidyl transferase mediated dUTP Nick End Labeling)检测了文蛤各发育阶段的幼虫中细胞凋亡的部位,利用Caspase原位标记分析了有活性的Caspase在幼虫中的分布情况,用Caspase抑制剂Z-VAD-FMK处理受精后4天和5天的变态前幼虫,同时以抑制剂的有机溶剂DMSO(Dimethyl sulfoxide)作对照,根据其对幼虫变态的影响,分析Caspase在文蛤幼虫变态中的作用.细胞凋亡检测结果显示,幼虫发育过程中发生细胞凋亡的部位主要在壳顶幼虫的面盘,说明细胞凋亡可能是文蛤幼虫变态过程中面盘退化的主要机制;在担轮幼虫至壳顶幼虫中都检测到有活性的Caspase,说明整个幼虫形态变化过程中都有Caspase的参与;用Caspase特异性抑制剂处理变态前幼虫,发现处理组幼虫变态率均显著低于对照组(P<0.05),说明Caspase在变态早期和晚期都有重要作用,同时发现DMSO对幼虫变态有诱导作用,可以在生产中加以利用.

APOPTOSIS AND ROLE OF CASPASE IN DEVELOPMENT OF MERETRIX MERETRIX LARVAE

Apoptosis plays an important role in larval development of species. Taking clam Meretrix meretrix as an example, to the apoptosis and function of caspase in bivalve larvae development and metamorphosis were studied. Apoptosis was detected in larvae at different stages with TUNEL labeling (Terminal-deoxynucleotidyl transferase mediated dUTP Nick End Labeling). The distribution of activated caspase in larvae was examined with CaspACE^TM FITC-VAD-FMK In Situ Marker. The pan-caspase inhibitor (Z-VAD-FMK) was used to analyze the function of caspase in 4-day-old and 5-day-old larvae post fertilization. Dimethyl sulfoxide (DMSO), the solvent of the inhibitor, was added to seawater as control. According to the effect on metamorphosis, the function of caspases in larvae was analyzed. The results reveal that the main apoptosis event occurred in the velum of pediveliger before metamorphosis, indicating that the degradation of velum might be the process of apoptosis. The activated caspases were observed in the larvae from trochophore to pediveliger, suggesting the caspases are involved in the whole developmental process. Having detected no signal in the velum, we proposed that the apoptosis in velum might be caspase-independent. The 4-day-old and 5-day-old larvae under treatment of caspase inhibitor showed significant lower metamorphosis rates than that in the DMSO control group (P<0.05). The results indicate that the caspases had functioned in early and later stages of metamorphosis, and also that DMSO could be an inducer in larvae metamorphosis.