AMPK调控内皮祖细胞参与子宫－胎盘循环建立在子痫前期发生中的作用及机制研究

Placental ischemia is the key event in the pathogenesis of preeclampsia (PE). On one hand, compromised invasiveness of extravillous cytotrophoblastic cells (EVCT) contributes to insufficient replacenment of original conpomemts of spiral arteries, on the other hand, fail to repair EVCT invasion caused vascular injury lead to intimal hyperplasia and loss of vasodialationm, both of which may also results in insufficient utero-placental perfusion. Endothelial progenitor cells（EPC) are critical vascular protective factor that involved in angiogenesis and maintaining structural integrity of blood vessels, while AMP-activated protein kinase (AMPK) is the pivotal regulator of metabolism, which has profound influence in cellular physiological functions. Our preliminary data has shown that the number and function of circulating EPC from PE patients are significantly different from normal controls, in addition to significantly compromised AMPK phosphorylation. In vitro experiments confirmed that AMPK is involved in regulating the angiogenic function of EPC. Therefore, this study proposes that AMPK is the key regulator of EPC’s vascular repair function and thus play an import role in the establishment of utero-placental circulation during early pregnancy. The abnormal decrease in AMPK activity of EPC could be a causative factor for PE development. This study plan to target at EPC, and investigate the molecular regulatory mechanism of EPC mediated vascular repair function by AMPK, which might provide novel strategy and experimental evidence for PE prevention and management.

胎盘缺血是子痫前期（PE)发生的关键因素，一方面绒毛外滋养细胞(EVCTs)侵袭力下降可造成子宫螺旋动脉重铸不足,另一方面EVCTs侵袭造成的内皮损伤未能有效修复导致内膜增生和舒张功能障碍也可能是胎盘灌注不足的重要原因。内皮祖细胞（EPCs）是调控血管生成、维持血管稳定性的重要血管保护因子,而腺苷酸活化蛋白激酶(AMPK)是能量代谢的关键酶,对细胞生理功能具有重要调控作用。本课题组发现PE病人外周血EPCs功能出现异常，同时AMPK磷酸化水平显著下降，体外实验证实AMPK参与调控EPCs的血管生成功能。由此推测AMPK活性对于EPCs在子宫－胎盘循环建立过程中发挥血管损伤修复功能至关重要,EPCs的AMPK活性下降可能是导致PE发生的重要原因之一。本研究拟以EPCs为研究对象，深入探讨AMPK调控EPCs血管损伤修复功能的分子机制,有望为PE防治提供新的思路和实验证据。

胎盘缺血是子痫前期（PE)发生的关键因素，一方面绒毛外滋养细胞(EVCTs)侵袭力下降可造成子宫螺旋动脉重铸不足,另一方面EVCTs侵袭造成的内皮损伤未能有效修复导致内膜增生和舒张功能障碍也可能是胎盘灌注不足的重要原因。内皮祖细胞（EPCs）是调控血管生成、维持血管稳定性的重要血管保护因子,而腺苷酸活化蛋白激酶(AMPK)是能量代谢的关键酶,对细胞生理功能具有重要调控作用。.本项目发现PE病人外周血EPCs（PEPCs）相较于正常足月妊娠外周血EPCs（NEPCs）而言，细胞集落形成和促血管形成能力均显著降低，同时AMPK磷酸化水平显著下降。对利用SCID免疫缺陷小鼠建立颈总动脉血管损伤模型注射PEPCs和NEPCs， Myograph结果显示PEPCs注射后血管非内皮依赖性血管舒张功能显著降低。Masson染色和OPN染色，结果显示PEPCs注射后的血管周围出现了显著的胶原累积。.经尾静脉注射PEPCs至孕鼠体内，孕鼠出现子痫前期样表型：Myograph结果显示PEPCs注射引起孕鼠子宫动脉舒张功能显著降低，子宫-胎盘切片染色结果显示，PEPCs引起子宫螺旋动脉周围胶原沉积增多。而在人蜕膜切片免疫组化染色结果显示PE中FN和Collagen Ia高表达，Masson染色提示PE蜕膜中胶原纤维增多，以上结果提示PE子宫螺旋动脉周围胶原沉积增多。对正常妊娠和PE胎膜进行10X genomics RNA-seq，发现PE蜕膜中VSMCs高表达ECM相关基因，合成型VSMCs比例高于正常妊娠。.利用蛋白芯片检测PEPCs和NEPCs培养基，发现sEndoglin在PEPCs培养基中升高，而PEPCs中HO-1蛋白水平低于NEPCs，在NEPCs中敲除AMPKα1，HO-1蛋白水平比对照组有明显降低。ELLISA结果显示过表达HO-1的PEPCs培养基中sEndoglin减少，但经HO-1抑制剂Znppix处理后sEndoglin水平得以回复。HO-1抑制剂Znppix处理NEPCs后显著抑制共培养模型的血管形成过程，而HO-1激动剂Hemin处理PEPCs后增强共培养模型的血管形成过程。.以上研究结果表明AMPK通过HO-1负性调节sEndoglin从而调控EPCs的血管内皮再生功能,EPCs的AMPK活性下降可能是导致PE发生的重要原因之一。