【作者】 刘静；

【导师】 孟金来；

【作者基本信息】 山东大学 ， 妇产科学（专业学位）， 2022， 硕士

【摘要】 背景子痫前期(pre-eclampsia,PE)是一种严重威胁母儿健康的妊娠并发症,其发病机制尚未完全明确。胎盘滋养细胞浸润不足可造成流产、子痫前期等妊娠期疾病,而侵袭过度则会导致绒毛膜癌等疾病的发生。Gastrokine 1(GKN1),也称为AMP18(18 kDa胃窦黏膜蛋白),是一种分泌蛋白,表达于胃窦和胃底表面黏膜细胞,通过调节增殖和分化抑制肿瘤侵袭,并通过诱导特定信号通路介导细胞凋亡。目前的研究集中在探索GKN1的肿瘤抑制特性及其在胃肠道肿瘤疾病治疗中的潜在临床应用,对GKN1在其他器官系统中的表达和功能研究甚少。有研究表明GKN1在正常妊娠中的胎盘以及妊娠滋养层疾病中表达,并且GKN1定位于胎盘绒毛外滋养细胞。胎盘组织基因阵列分析表明GKN1在胎盘中的表达水平相当高。目的研究正常妊娠和子痫前期胎盘组织中GKN1 mRNA及蛋白质的表达情况;利用体外细胞学实验的方法研究GKN1对子痫前期患者胎盘滋养细胞迁移、侵袭和增殖的影响;通过GKN1与JAK-STAT信号通路的关系研究了解其发挥作用的生物学机理;旨在探究子痫前期的发病机制加速疾病的治疗进展。方法1.收集正常妊娠和子痫前期患者胎盘组织各30例,Western blot检测GKN1蛋白的表达;免疫组化检测胎盘组织中GKN1的表达;qRT-PCR检测GKN1 mRNA的表达。2.低氧1%环境下体外模拟子痫前期培养HTR-8/Svneo细胞,并通过qPCR以及Western blot检测HTR-8/Svneo细胞中GKN1的表达变化来进行验证。3.通过小干扰RNA对低氧1%环境下培养的HTR-8/Svneo细胞进行转染处理来敲降GKN1。然后进行MTT实验和EdU实验检测细胞增殖能力,划痕实验检测对迁移的影响,Transwell检测侵袭能力。4.Western blot检测敲降 GKN1 对低氧条件下 HTR-8/Svneo细胞中 p-JAK2、JAK2、p-STAT3和STAT3的表达变化的影响。结果1.子痫前期患者胎盘组织GKN1 mRNA及蛋白表达水平高于正常妊娠组,差异有统计学意义(P<0.01)。免疫组织化学结果显示,子痫前期患者的胎盘组织中GKN1的表达增加。2.HTR-8/SVneo细胞在低氧条件下培养,GKN1的mRNA蛋白表达水平均较正常培养状态显著增加(P<0.01)。3.低氧条件下GKN1敲低导致HTR-8/SVneo细胞的迁移能力增加,增殖和侵袭能力显著加强。4.GKN1敲低时低氧条件下的HTR-8/Svneo细胞中p-JAK2和p-STAT3蛋白质表达水平增加,JAK-STAT信号通路被激活。结论1.子痫前期患者胎盘中GKN1在翻译及转录水平表达增高,推测GKN1可能参与了子痫前期的发病机制。2.GKN1抑制滋养细胞的增殖、迁移以及侵袭,GKN1参与子痫前期的发病可能是通过胎盘浅植入来完成。3.GKN1可能通过调控JAK-STAT信号通路参与子痫前期的发病。更多还原

【Abstract】 BackgroundThe pre-eclampsia(PE)is a serious pregnancy complication to the health of mother and child,its pathogenesis is still incompletely clear.Insufficient infiltration can cause pregnancy diseases such as abortion and preeclampsia.On the contrary excessive invasion of placental trophoblasts can lead to the occurrence of diseases such as choriocarcinoma.Gastrokine 1(GKN1),also known as AMP 18(18 kDa antral mucosal protein),is a secreted protein expressed in mucosal cells on the surface of gastric antral and fundus,which suppresses tumor invasion by regulating proliferation and differentiation and induce signaling pathways to mediate apoptosis.The current study has focused on exploring the tumor suppressor properties of GKN1 and its potential clinical application in the treatment of gastrointestinal tumor diseases,with little research on the expression and function of GKN1 in other organ systems.Studies have shown that GKN1 is expressed in the placenta of normal pregnancy as well as gestational trophoblast diseases,and GKN1 located in extravillous trophoblast(EVT)of placenta.Gene array analysis in the placental tissue showed that the expression level of GKN1 in the placenta is quite high.ObjectiveTo study the expression of GKN1 mRNA and protein in the placenta of normal pregnancy and preeclampsia;Explore the effect of GKN1 on invasion、migration and proliferation of trophoblast cells through vitro cytology experiments;Rearch the biological mechanism of GKN1 and JAK-STAT signaling;Provide new ideas for the study of pathogenesis and early diagnosis as well as effective treatment of preeclampsia.Methods1.Thirty cases of normal pregnancy and preeclamptic placental tissue were collected to test GKN1 protein expression by Western blot,GKN1 mRNA expression by qRT-PCRand GKN1 expression by immunohistochemistry.2..HTR-8/Svneo cells were cultured in 1%low oxygen conditions in order to mimic preeclampsia in vitro and we verified it by qPCR and Western blot for GKN1 expression changes in HTR-8/Svneo cells.3.First of all,GKN1 was knocked down after transfection by siRNA of HTR-8/Svneo cells,then we put cells cultured in 1%low-oxygen conditions.Finally we test the proliferation ability by MTT and EDU,the invasion ability by Transwell,and the migration ability of Scratch test.4.The effect of knockdown of GKN1 on the expression changes of p-JAK2,JAK2,p-STAT3,and STAT3,in the hypoxia model of HTR-8/Svneo cells was determined by Western blot.Results1.Compared with normal pregnancy,the GKN1 mRNA and protein expression level of preeclamptic placenta tissue increased significantly,with a significant difference(P<0.01).Immunohistochemical results showed an increased expression of GKN1 in placental tissue of pre-eclampsia.2.When HTR-8/SVneo cells were cultured under hypoxiaboth the mRNA and protein expression levels of GKN1 significantly increased(P<0.01).3.GKN1 knockdown under hypoxia caused increased proliferative capacity of HTR-8/SVneo cells,both migration and invasion significantly enhanced.4.When GKN1 was knocked down in the HTR-8/Svneo cell hypoxia model,the expression levels of the p-JAK2 and p-STAT3 proteins were increased,and the JAK-STAT signaling pathway was activated.Conclusions1.GKN1 expression is increased in preeclampsia,we speculated that GKN1 may be involved in the pathogenesis of preeclampsia.2.GKN1 has inhibitory effects on the proliferation,migration and invasion of trophoblast cells,it may involved in the pathogenesis of preeclampsia by causing superficial placental implantation.3.GKN1 may participate in the pathogenesis of preeclampsia by regulating the JAK-STAT signaling pathway.更多还原