家蚕幼虫-蛹变态期前胸腺和脂肪体细胞解离和程序性细胞死亡的比较分析  

Comparative analysis of cell dissociation and programmed cell death in the prothoracic gland and fat body of Bombyx mori during the laval-pupal metamorphosis

作　　者：龙诗慧 李瑜 田铃[2] 李胜 李康[1] LONG Shi-Hui;LI Yu;TIAN Ling;LI Sheng;LI Kang(Guangdong Provincial Key Laboratory of Insect Development Biology and Applied Technology/Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China;Key Laboratory of Agro-animal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Science, South China Agricultural University, Guangzhou 510642, China)

机构地区：[1]华南师范大学生命科学学院,广东省昆虫发育生物与应用技术重点实验室/昆虫科学与技术研究所,广州510631 [2]华南农业大学动物科学学院,广东省农业动物功能基因组学与分子育种重点实验室/广东省蚕桑工程技术研究中心,广州510642

出　　处：《昆虫学报》2019年第11期1239-1249,共11页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31702053)

摘　　要：【目的】检测家蚕Bombyx mori变态期前胸腺细胞的解离、自噬与凋亡,并与脂肪体的进行对比,从而解析昆虫幼虫-蛹变态期过程中不同组织重塑的异同。【方法】以家蚕5龄期、游走期、预蛹期和蛹期前胸腺和脂肪体组织为材料,在光学显微镜下观察前胸腺和脂肪体细胞解离情况;分别利用Lyso-Tracker和TUNEL染色,在荧光共聚焦显微镜下观察细胞自噬和细胞凋亡的发生情况;利用qRT-PCR检测家蚕前胸腺中自噬发生标志基因Atg8的表达水平;利用透射电镜观察前胸腺和脂肪体细胞自噬小体和前胸腺线粒体;利用Caspase3酶活性检测试剂盒测定Caspase3酶活性;利用qRT-PCR检测前胸腺中蜕皮酮(ecdysone)合成相关基因Spo,Phm,Dib和Sad的表达水平;利用酶免疫试验(enzyme-immunoassay,EIA)测定前胸腺中蜕皮酮的含量,进而检测合成蜕皮酮的活力。【结果】在家蚕幼虫到蛹的变态发育过程中,在化蛹第1天家蚕前胸腺和脂肪体细胞中同时开始出现细胞解离;脂肪体细胞自噬和凋亡分别在游走期和预蛹第1天开始出现并逐渐增强;而前胸腺一直到化蛹第2天都没有发生明显的细胞自噬和凋亡;此外,前胸腺中线粒体的形态变化和蜕皮酮合成相关基因的转录水平均与对应时期前胸腺合成蜕皮酮的活力一致。【结论】在变态发育时家蚕不同组织消亡发生的时间不同,虽然前胸腺和脂肪体在化蛹第1天同时出现细胞解离,但是前胸腺直到化蛹第2天都不发生细胞自噬和凋亡,可能与其持续合成蜕皮酮的功能有关。本研究为昆虫幼虫-蛹变态发育时期组织消亡的深入研究提供了理论依据与工作基础。【Aim】To detect the cell dissociation,autophagy and apoptosis in the prothoracic gland of the silkworm,Bombyx mori during metamorphosis,and to compare tissue demise in the prothoracic gland with that in the fat body,so as to uncover the similarities and differences of tissue remodeling during the larval-pupal metamorphosis.【Methods】With the prothoracic gland and fat body tissues of B.mori at the 5th instar,wandering,prepupal and pupal stages as the test materials,the cell dissociation in the prothoracic gland and fat body of B.mori was observed under light microscopy.The autophagy and apoptosis were observed after Lyso-Tracker and TUNEL staining,respectively,under fluorescence confocal microscopy.The expression profile of the autophagy marker gene Atg8 in the prothoracic gland was detected by qRT-PCR.The autophagosome in the prothoracic gland and fat body as well as the morphological changes of mitochondria in the prothoracic gland were observed under transmission electron microscopy.The Caspase3 activity was assayed using Capase3 enzyme assay kit.The expression profiles of Halloween genes Spo,Phm,Dib and Sad were detected by qRT-PCR.The ecdysone content in the prothoracic gland was detected by enzyme-immunoassay(EIA)aiming at furthermore determining the activity of the synthesis of ecdysone.【Results】During the transition from larva to pupa of B.mori,both the prothoracic gland and fat body began to undergo cell dissociation on the 1st day of pupation.Autophagy and apoptosis in the fat body began to appear at the wandering stage and day 1 of prepupa,respectively,and gradually increased.However,autophagy and apoptosis did not occur in the prothoracic gland until the 2nd day after pupation.In addition,the morphological changes of mitochondria and the transcription levels of Halloween genes were consistent with the activity of ecdysone synthesis in the prothoracic gland at the corresponding developmental stage of B.mori.【Conclusion】The time of tissue demise is different in different tissues of B.mori d

关 键 词：家蚕 前胸腺 脂肪体 细胞解离 自噬 凋亡 程序性细胞死亡 

分 类 号：S881[农业科学—特种经济动物饲养]