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目的:探讨高浓度葡萄糖对小鼠囊胚细胞线粒体和细胞色素氧化酶的影响,探讨高浓度葡萄糖对小鼠囊胚有氧代谢的影响,为糖尿病妊娠导致胎儿畸形的机制和预防提供理论依据。方法:通过促超排卵,获取妊娠3.5天小鼠囊胚,随机分成三组,即对照组(空白)、低糖组(葡萄糖浓度为7.5 mmol/L)和高糖组(葡萄糖浓度为28.0 mmol/L),分别培养在含0、7.5 mmol/L和28.0 mmol/L葡萄糖的M199培养基中,培养24 h后,然后再吸山囊胚。每组随机吸取30个囊胚用罗丹明123荧光染料标记线粒体和组织化学方法显示细胞色素C氧化酶,观察囊胚细胞线粒体和细胞色素C氧化酶(Cytochrome C Oxidase)的变化。结果:①高糖组小鼠囊胚线粒体功能活性明显降低,荧光密度值较低,且线粒体的位置分布发生了明显变化,空白组和低糖组囊胚胞浆中线粒体分布均匀,荧光密度值较高,线粒体仍具有一定的活性。空白组与低糖组囊胚荧光密度值无显著性差异(P>0.05),高糖组与空白组和低糖组相比均存在显著性差异(P<0.05);②细胞色素氧化酶(COX)活性的变化:高糖组小鼠囊胚胞浆淡染,反应产物呈浅黄色,COX活性明显降低;空白组和低糖组囊胚胞浆可见明显的棕褐色物质,COX活性较高,统计学分析,空白组与低糖组囊胚灰度值无显著性差异(P>0.05),高糖组与空白组和低糖组相比均存在显著性差异(P<0.05)。结论:高浓度葡萄糖对小鼠囊胚线粒体的分布、功能以及COX活性均有影响。
OBJECTIVE: To investigate the effects of high glucose on mitochondria and cytochrome oxidase in mouse blastocysts, and to explore the effect of high glucose on the aerobic metabolism in mouse blastocysts, providing a theoretical basis for the mechanism and prevention of fetal malformations in diabetes. Methods: The blastocysts of 3.5 days gestation were obtained and the control group (blank), low glucose group (7.5 mmol / L glucose) and high glucose group (glucose concentration 28.0 mmol / L) were cultured in M199 medium containing 0, 7.5 mmol / L and 28.0 mmol / L glucose, respectively, and then cultured for 24 h. 30 blastocysts were randomly selected from each group. Cytochrome C oxidase was detected by mitochondrial and histochemical staining with rhodamine 123 fluorescent dye, and the changes of mitochondria and cytochrome C oxidase in blastocysts were observed. Results: ①The functional activity of mitochondria of blastocyst in high glucose group was significantly decreased, the fluorescence density value was lower, and the mitochondria ’s position distribution changed obviously. The distribution of mitochondria in cytoplasm of blank group and low glucose group was uniform, High, mitochondria still have some activity. (P> 0.05). There was significant difference between high glucose group and blank group and low glucose group (P <0.05). ② The levels of cytochrome oxidase (COX) Changes in activity: In the high glucose group, the cytoplasm of the mouse blastocyst was lightly stained, the reaction product was light yellow, and the COX activity was significantly decreased. The cytoplasm of the blastocyst in the blank group and the low glucose group showed obvious tan substance with higher COX activity There was no significant difference (P> 0.05) between the gray level of blastocysts of the blank group and the low glucose group, and there was significant difference between the high glucose group and the blank group and the low glucose group (P <0.05). Conclusion: High concentration of glucose affects the distribution, function and COX activity of mouse blastocyst mitochondria.