热应激对小鼠胚胎细胞HSP70表达和超微结构的影响

Effect of Heat Shock on HSP 70 Expression and Ultrastructure of Mouse Embryonic Cell

本研究旨在确定体外培养的小鼠胚胎细胞合成HSP70的时期,研究热应激对该阶段胚胎细胞超微结构的影响。将合子和体外发育至2-细胞、4-细胞、8～16-细胞及囊胚阶段的胚胎分别进行37、39和41℃的热应激处理,用免疫荧光细胞化学法检测胚胎细胞中HSP70的诱导表达;将体外培养至早期囊胚的小鼠胚胎分别施以39℃2h和41℃2h处理,分别在热应激后0和2h时收集胚胎,制作超薄切片,用电子显微镜观察胚胎细胞的超微结构变化。在各细胞期胚胎中,37℃组胚胎HSP70表达阴性(-);39℃组胚胎,从8～16-细胞开始呈现弱表达(+),囊胚呈阳性表达(++);41℃组的胚胎从4-细胞开始呈现弱阳性表达(+),8～16-细胞胚胎和囊胚呈阳性表达(++)。39和41℃热应激处理的小鼠早期囊胚超微结构和对照组均发生明显的变化,但经37℃恢复培养2h后,39℃组胚胎的各细胞器超微结构恢复正常,而41℃组胚胎未能恢复。结果表明,小鼠胚胎从4-细胞期有HSP70的诱导合成,在囊胚期诱导合成最多;热应激使胚胎亚细胞结构出现退行性变化,轻度退行性变化是可逆的。

The aims of this study were to determine the developmental stage when HSP 70 was synthesized in the mouse embryo induced by heat shock in vitro and to study the effect of heat shock on the ultrastructural changes of the embryo in the corresponding period.The zygotes and embryoes at the 2-cell,4-cell,8-16-cell and blastular stages were treated with heat stress at the temperature of 37,39 and 41 ℃,respectively,to determine the expression of HSP70 by the method of immunofluorescence and cytochemistry.The mouse embryos at the early blastular stage were treated separately in vitro at 39 and 41 ℃ for 2 h.Then the embryos were collected at 0 and 2 h after heat shock to make ultrathin section;the transmission electronic microscope was used to observe the change of embryo ultrastructure.The expression of HSP70 was different at different embryonic development stages.There was a negative（-） expression in the 37 ℃ group of all stage embryo.However,in the 39 ℃ group of embryos,there was a weak expression（＋） of HSP70 in the 8-16-cell stage and a positive expression（＋＋） at the blastular stage.In the 41 ℃ group,HSP70 was weakly expressed（＋） at the 4-cell stages and positively expressed（＋＋） both at 8-16-cell and blastular stages.There was a significant change in the ultrastructure of early blastula induced by both 39 and 41 ℃ heat shock compared to the control group.The ultrastructural change of embryo organelle treated in 39 ℃ group recovered to normal by culture at 37 ℃ for 2 h.However,the recovery was not occurred in the embryos treated at 41 ℃.These results indicate that the HSP70 is induced to synthesize in the embryos from 4-cell stage and is mostly synthesized at the blastular stage;heat shock changes subcellular structure of the embryo,which appear to have degenerative changes,however,the mild changes are reversible.