家蚕翅原基细胞的凋亡和自噬分析  

Analysis of apoptosis and autophagy in wing discs of the silkworm,Bombyx mori

作　　者：刘学术[1] 胡启豪 郑思春[1] 冯启理[1] 邓惠敏[1] 

机构地区：[1]华南师范大学生命科学学院,广州市昆虫发育调控与应用重点实验室,广州510631

出　　处：《昆虫学报》2016年第12期1317-1324,共8页Acta Entomologica Sinica

基　　金：国家自然科学基金重点项目(31330071);国家自然科学基金青年基金项目(31301918)

摘　　要：【目的】家蚕Bombyx mori翅原基在生长分化过程中伴随着造血器官和翅囊等组织的消失。本研究旨在分析家蚕翅原基生长分化过程中是否存在细胞凋亡和细胞自噬。【方法】制作蛹期0 d翅原基石蜡切片,利用TUNEL法检测细胞凋亡情况;提取5龄第3天、5龄第6天、上簇第1天、预蛹期、蛹期0 d及蛹期第3天的翅原基总RNA,反转录成c DNA,利用定量PCR检测凋亡和自噬相关基因的表达水平;检测了细胞凋亡相关的Caspase 3和细胞自噬相关的酸性磷酸酶活性。【结果】TUNEL染色发现蛹期0 d的翅原基出现部分细胞凋亡现象;细胞凋亡相关基因Caspase 3和Nc主要在5龄幼虫期有高水平表达,细胞凋亡抑制因子IAP主要在幼虫末期有较高水平的表达;Caspase 3酶活性在上簇第1天出现峰值;细胞自噬相关基因Atg5,Atg6,Atg8和Atg12主要在幼虫末期有较高水平的表达;酸性磷酸酶活性也主要在幼虫末期较高。【结论】在幼虫期可能存在细胞凋亡,抑制了翅原基细胞的增殖,进而阻止了翅原基的生长分化;在幼虫末期可能同时存在细胞凋亡和自噬,促进翅囊等的退化,同时阻止了翅原基细胞的增殖,推动了翅原基向蛹翅的发育。【Aim】 The process of the growth and differentiation of wing discs of the silkworm, Bombyx mori is accompanied with the disappearance of such tissues as hematopoietic organs and wing bursa. The study aims to analyze whether apoptosis and autophagy occur during the growth and differentiation of wing discs in the silkworm. 【Methods】 Apoptosis in wing discs of B. mori at the pupal stage was analyzed by TUNEL. The total RNA of wing discs at day-3 and day-6 5th instar larval stage, day-1 wandering stage, pre-pupal stage, and day-0 and day-3 pupal stage were extracted and then reversely transcribed into cDNA. The expression patterns of apoptosis and autophagy related genes were detected by qRT-PCR. The activities of Caspase 3 and acid phosphatase were analyzed. 【Results】 It was observed that apoptosis occurs in the partial wing discs cells at day-0 pupal stage. The apoptosis related genes Caspase 3 and Nc had higher expression levels during the 5th instar larval stage, while IAP had higher expression level at the late larval stage than at the pupal stage. The activity of Caspase 3 reached the peak at day-1 wandering stage. The autophagy related genes Atg5, Atg6, Atg8 and Atg12 had higher expression levels during the late larval stage. The acid phosphatase activity was higher during the late larval stage. 【Conclusion】 Apoptosis might occur during the larval stage to inhibit the cell proliferation of wing discs and to prevent wing discs from growth and differentiation. Apoptosis and autophagy might occur at the late larval stage to promote the degeneration of wing bursa, to inhibit the cell proliferation of wing discs, and to push forward the development of pupal wing from wing discs.

关 键 词：家蚕 翅原基 凋亡 自噬 CASPASE 3 酸性磷酸酶 

分 类 号：Q966[生物学—昆虫学]