miR-1和miR-21在先兆子痫发病早期AT1受体自身抗体诱导心肌细胞凋亡中作用及机制研究

These observations have been supported by a large study showing that women with a history of preeclampsia/eclampsia have high susceptibility of the myocardial to ischemic damage, approximately double the risk of early cardiac, cerebrovascular and peripheral arterial diseases and cardiovascular mortality compared to women with uncomplicated pregnancies even after controlling for many confounders. Recently we found that the agonistic autoantibodies to AT1 receptor (AT1-AA) can cause the apoptosis of cardiomyocytes in pregnant rats, resulting in the changes of cardiac structure and function, and occur preeclampsia and increased susceptibility to ischemic injury of postpartum heart. miR-1 and miR-21 is not only highly expressed in the cardiovascular system，but also be aberrantly expressed in many cardiovascular diseases, such as myocardial infract, heart failure and cardiac hypertrophy, plays an important role in the pathophysiological and molecular mechanism of various cardiovascular diseases. miR-1 and miR-21 has anti-apoptotic effects, the abnormal expression of miR-1 and miR-21 is involved in the pathogenesis of preeclampsia of cardiomyocytes apoptosis is not clear. This research adopts the modern molecular biology techniques, to explore the: the change of myocardial tissue miR-1 and miR-21 in preeclampsia model induced by AT1-AA; miR-1 and miR-21 genomic sequence was amplified by PCR and cloned into lentivirus vector, an eukaryotic expression vector for lenti-miR-1 or lenti-miR-21 was constructed. After transferred lenti-miR-1 or lenti-miR-21 into the pregnant rats via tail vein injectionm, to observe the over expression of miR-1 and miR-21 can inhibit the cardiomyocytes apoptosis whether preeclampsia rat. In vitro experiment：neonatal rat cardiomyocytes were transfeeted with miR-1 and miR-21 inhibitors to determined by the cardiomyocytes apoptotic index to evaluate the effect of miR-1 and on AT1-AA-induced cardiomyocytes apoptosis. miR-1 and miR-21 and PTEN mRNA were examined by qRT-PCR. Intracellular signal molecules, such as the expression of PTEN, phosphorylated PTEN, AKT, phosphorylated Akt, FOXO3a, phosphorylated FOXO3a were detected by Western blot. Through this research reveals that the role of miR-1 and miR-21 in the cardiomyocytes apoptosis in preeclampsia model. For future interventions, miR-1 and miR-21 provides a new target for therapy for cardiovascular diseases.

近年来研究发现AT1受体自身抗体(AT1-AA)与先兆子痫发病密切相关。我们通过系列研究发现AT1-AA诱导心肌细胞凋亡参与了先兆子痫的发病过程。最近我们对AT1-AA处理后的乳鼠心肌细胞miRNA表达谱进行筛选时发现miR-1和miR-21表达下调，而miRNA表达异常与许多疾病发生有关。本研究拟用AT1-AA复制先兆子痫大鼠模型，证实miR-1和miR-21在AT1-AA诱发先兆子痫过程中被抑制；通过在体过表达miR-1和miR-21，确认它们作为治疗先兆子痫新靶点的可能性；利用培养的乳鼠心肌细胞，探讨AT1-AA如何抑制miR-1和miR-21表达、PTEN是否为miR-1和miR-21靶基因、PTEN/AKT/FOXO3a信号转导通路是否参与了miR-1和miR-21抑制心肌细胞凋亡过程？本研究将进一步阐述AT1-AA在先兆子痫发病中的作用，为治疗先兆子痫等疾病提供新思路。

miR-1和miR-21在先兆子痫发病早期AT1受体自身抗体诱导心肌细胞凋亡中作用及机制研究..背景：心肌细胞凋亡是多种心血管疾病的重要病理生理机制之一。在先兆子痫患者血浆中存在的AT1-AA可引起心肌细胞凋亡。近年来研究发现AT1-AA与先兆子痫发病密切相关。我们通过系列研究发现AT1-AA诱导心肌细胞凋亡参与了先兆子痫的发病过程。miR-1和miR-21在心血管系统中呈高表达，对心肌细胞发挥保护作用，而PTEN/AKT/ FOXO3a通路是miR-21介导的心血管效应的靶基因。本研究的目的是分析在用AT1-AA复制先兆子痫大鼠模型过程中，miR-1和miR-21的表达是不是被抑制。通过在体过表达miR-1和miR-21，确认它们作为治疗先兆子痫新靶点的可能性。..研究内容：用从先兆子痫病人血浆中提取的 AT1-AA 建立先兆子痫大鼠模型，观察 miR-1和miR-21 在大鼠心肌的表达情况；分析先兆子痫大鼠心肌细胞凋亡与 miR-1 和miR-21 水平之间的关系；构建 miR-1 和miR-21 慢病毒表达载体转染动物，分析miR-1 和miR-21 作为先兆子痫治疗靶点的可能性；同时进一步分析miR-1 和miR-21 对AT1-AA 诱导的心肌细胞凋亡的信号通路，确认PTEN/AKT/FOXO3a信号转导通路是否参与 miR-1 和 miR-21 对 AT1-AA 诱导的心肌细胞凋亡的作用。.研究结果：应用qRT-PCR法检测发现AT1-AA能明显降低miR-1和miR-21的表达而增加PTEN mRNA的表达，PTEN mRNA的表达变化趋势与miR-1和miR-21的表达变化趋势相反，结果提示在基因水平上，PTEN可能为miR-21对AT1-AA诱导的心肌细胞凋亡的影响中的靶基因之一。与正常心肌细胞组相比，转染了过表达miR-21的重组慢病毒载体的心肌细胞组中的PTEN表达下调，pPTEN，pAKTser473，pFOXO3a和FasL的表达上调。确定PTEN/AKT/FOXO3a 信号转导通路参与miR-1和miR-21对AT1-AA诱导的心肌细胞凋亡的作用。..科学意义：课题揭示miR-1和miR-21表达参与AT1-AA促进心肌细胞凋亡的过程，通过表达miR-1和miR-21重组慢病毒载体能够抑制AT1-AA诱导的心肌细胞凋亡，该作用可能是通过介