脂联素通过AdipoR2-AMPK-内质网应激-凋亡途径对高糖环境下滋养细胞损伤的保护作用及临床意义

This study is based on the theory of the excessive apoptosis in placental cytotrophoblasts of gestational diabetes mellitus and antiapoptosis property of adiponectin. The experiments focus on identifying the mechanism how adiponectin protects injured cytotrophoblasts under high glucose environment through AdipoR2 - AMPK - endoplasmic reticulum stress response - apoptosis pathway by researching on the effect of adiponectin on the apoptosis of cytotrophoblasts from pregnant women with gestational diabetes mellitus and cell line Bewo cultured in vitro. The steps of this study are designed as below: (1) Culture the cytotrophoblast cell line Bewo under high glucose environment to build up the cell model. Observe the changes of endoplasmic reticulum stress and apoptosis of cytotrophoblast in placenta with gestational diabetes mellitus and under high glucose environment in vitro by transmission electron microscopy, flow cytometry instrument, RT-PCR and Western blot technique; (2) Clarify the association of the different endoplasmic reticulum stress and apoptosis with pregnancy outcomes in gestational diabetes mellitus; Clarify how adiponectin protects the cytotrophoblast by reducing the endoplasmic reticulum stress response and slowing down the apoptosis process; (4) AMPK inhibitor AraA and siRNA to suppress AdipoR2 are utilized, to identify if adiponectin activates the intracellular second messenger AMPK through AdipoR2 to participate in endoplasmic reticulum stress to regulate the apoptosis of cytotrophoblast. Thus, the results will be used to monitor and improve pregnancy outcomes of gestational diabetes mellitus, and lay a foundation of eugenic and superior nurture in our country.

本研究以妊娠期糖尿病胎盘滋养细胞存在过度凋亡和脂联素的抗凋亡作用为切入点，通过研究脂联素对妊娠期糖尿病孕妇胎盘滋养细胞及体外滋养细胞系Bewo细胞凋亡的影响，探讨脂联素通过AdipoR2-AMPK-内质网应激-凋亡途径对高糖环境下滋养细胞损伤的保护作用。（1）建立滋养细胞系Bewo高糖细胞模型，采用透射电镜、流式细胞仪、RT-PCR和Western blot技术检测孕妇及体外滋养细胞的内质网应激及凋亡情况；（2）观察分析妊娠期糖尿病胎盘滋养细胞内质网应激、凋亡与妊娠结局的关系；（3）探讨脂联素通过减轻内质网应激减轻滋养细胞凋亡，对滋养细胞起保护作用；（4）给予AMPK抑制剂阿糖腺苷(AraA）和小的干扰RNA抑制AdipoR2，探讨脂联素通过脂联素受体R2激活细胞内第二信使AMPK，让它参与内质网应激调控滋养细胞凋亡；最终为监测和改善妊娠期糖尿病妊娠结局提供有意义的实验依据和治疗的新靶点。

胎盘组织中滋养细胞增殖和凋亡的平衡在胎盘绒毛组织结构改建及功能的完善等方面发挥着重要的作用，对妊娠的维持和结局至关重要。在病理妊娠中，高糖、胎盘缺血等均可使滋养细胞增殖和凋亡失衡，导致妊娠结局不良。近年研究发现，脂联素具有调控内质网应激反应、抑制细胞增殖及抗凋亡双重功能，且与妊娠期糖尿病的发生、发展及妊娠结局密切相关。故研究脂联素对高糖环境下滋养细胞保护作用的具体机制对改善妊娠期糖尿病尤为重要。课题组在项目进行过程中发现：（1）妊娠期糖尿病及其妊娠结局与胎盘超微结构改变密切相关，孕期血糖控制情况、血清脂联素水平为妊娠期糖尿病孕妇胎盘超微结构改变的影响因素；（2）血清脂联素水平降低、血糖及胎盘脂联素受体2（AdipoR2）升高参与了妊娠期糖尿病合并巨大儿的形成，且妊娠期糖尿病合并巨大儿孕妇的胎盘滋养细胞内质网应激受到抑制，细胞增殖增加，妊娠结局不良；（3）高糖可导致滋养细胞BeWo的AdipoR2表达上调，抑制BeWo细胞的内质网应激反应而促进滋养细胞增殖，而脂联素可逆转这一现象；（4）通过检测高糖环境诱导滋养细胞增殖的具体机制通路，在此基础上，给予脂联素进行干预，并给予AdipoR2-AMPK-MEK-ERK信号通路中相关激酶的特异性抑制剂及激动剂，进行AdipoR2敲减，该部分研究发现脂联素可通过AdipoR2-AMPK-MEK-ERK-内质网应激途径抑制高糖环境下滋养细胞的增殖而起保护性作用。该研究将有助于指导临床寻找妊娠期糖尿病保护的新切入点，将为指导临床上如何改善妊娠期糖尿病妊娠结局提供有意义的实验依据和治疗的新靶点。