【作者】 林锟；

【导师】 林少达；

【作者基本信息】 汕头大学 ， 内科学， 2010， 硕士

【摘要】 背景与目的妊娠合并糖尿病发生率可达5%-20%,未及时诊断和治疗达标的妊娠合并糖尿病可引起巨大儿、胎儿畸形等并发症,并使流产、先兆子痫等发病率及母婴围生期死亡率增加,妊娠合并糖尿病患者自发性流产、胎儿重要器官先天性畸形发生率高达6%-10%。研究表明高血糖诱导的胚胎细胞凋亡是导致胎儿畸形和流产的主要机制之一。然而以往对高糖诱导胚胎凋亡的研究却局限于着床后胚胎和囊胚期。目前母体高血糖对卵母细胞和早期胚胎细胞凋亡的影响和机制尚未清楚。本研究拟构建STZ诱导糖尿病小鼠模型,检测卵母细胞、二细胞胚凋亡率和线粒体凋亡通路Bax、caspase-3表达水平,探讨母体高血糖对卵母细胞、二细胞胚胎凋亡的影响及其可能激活的Bax-caspase-3凋亡路径。材料与方法小鼠分为STZ诱导糖尿病组和非糖尿病组。超排卵取卵和二细胞胚。采用免疫荧光法检测卵母细胞和二细胞胚胎中Bax和caspase-3蛋白的表达;实时定量RT-PCR检测卵母细胞和二细胞胚胎中Bax和caspase-3 mRNA的表达;Annexin-V染色法检测卵母细胞和二细胞胚细胞凋亡;HE染色切片观察高血糖对卵巢内卵泡发育成熟的影响;电镜下观察高糖环境下二细胞胚超微结构改变。结果糖尿病组Annexin-V阳性(凋亡)卵母细胞明显多于非糖尿病组(P<0.001)。免疫荧光和实时定量RT-PCR结果显示:糖尿病组卵母细胞中Bax和caspase-3蛋白和mRNA表达水平均高于非糖尿病组(P<0.05)。HE染色切片结果显示:高血糖使卵泡生长延迟,生长期卵泡数量减少(P<0.05),且体积减小。在二细胞胚方面,无论在糖尿病组还是非糖尿病组,均未检测到Annexin-V阳性二细胞胚细胞。Bax蛋白和mRNA表达在糖尿病组二细胞胚中明显高于非糖尿病组(P<0.05),caspase-3蛋白和mRNA在两组中的表达则无差异(P>0.05)。电镜观察显示糖尿病组二细胞胚中可见大量明显肿胀线粒体。结论母体高血糖可能通过Bax-caspase-3途径诱导卵母细胞凋亡,导致卵泡生长发育迟缓。高血糖对二细胞胚同样有毒性作用但并非通过Bax-caspase-3途径实现。更多还原

【Abstract】 BACKGROUND AND OBJECTIVEThe ration of gestational diabetes to gestational women was up to 5%-20%, patients who were not diagnosised and managed in time had a higher incidence of large for date infant, fetal anomaly, preeclampsia, abortion and the incidence of spontaneous abortion and congenital malformation of vital organs was 6%-10%. It has been reported that hyperglycemia-induced inappropriate apoptosis in the developing embryos and oocytes may be one of the mechanisms leading to congenital malformation or miscarriage. The previous study was limited, however, and focused primarily on the effect of hyperglycemia on postimplantation embryo and blastocyst. Little is known about the effect of maternal diabetes on oocytes and earlier embryos apoptosis. The objective of this study was to investigate the occurrence of apoptosis and Bax and caspase-3 expression in oocytes and 2-cell embryos in streptozotocin(STZ)-induced diabetic mouse, examine the involvement of the Bax-regulated caspase-3 activation pathway in diabetes-induced apoptosis in oocytes and 2-cell embryos.METHODSAll mice were divided into 2 groups: STZ-induced diabetic group and nondiabetic group. Oocytes and 2-cell embryos were obtained by superovulation. Bax and Caspase-3 protein and mRNA were detected respectively by immunofluorescent and quantitative reverse transcription-polymerase chain reaction in oocytes and 2-cell embryos from diabetic vs nondiabetic mice. Apoptosis was detected by Annexin-V staining. Furthermore, HE-stained ovarian sections were made to see the effect of hyperglycemia on the oocytes maturation and development, electron microscopy was used to see the effect of hyperglycemia on the ultrastructure of 2-cell embryos.RESULTSThe increased number of Annexin-V-positive cells occurred in diabetic oocytes compared to non-diabetic oocytes(P<0.05). In quantitative RT-PCR and immunofluorescent, Bax and caspase-3 expression were significantly increased in diabetic oocytes than non-diabetic oocytes(P<0.05). HE-stained ovarian sections demonstrated that hyperglycemia resulted in delayed follicular growth as detailed by reduced number of growing follicles(P<0.05). In contrast, no Annexin-V-positive cell in 2-cell embryos was found in diabetic and non-diabetic mouse. Although Bax expression was elevated in diabetic 2-cell embryos(P<0.05), caspase-3 expression in 2-cell embryos was no significant difference between diabetic and non-diabetic mouse(P>0.05). Electron-Microscopy study revealed that more swollen mitochondrias were found in diabetic 2-cell embryos.CONCLUSIONSMaternal diabetes might increase oocyte apoptosis by a Bax-caspase-3 pathway to play a role in embryonic malformations by delayed oocyte development. Development of 2-cell embryos might be adversely affected by maternal diabetes, but not through Bax-regulated caspase-3 apoptotic pathway.更多还原