【作者】 李明辉；

【导师】 刘锦纷；

【作者基本信息】 上海交通大学 ， 外科学（心胸外科）， 2018， 博士

【摘要】 研究背景和目的:动脉导管依赖性先天性心脏病严重影响患儿的生存率,尤其是当动脉导管闭合后,患儿需在最短的时间内接受手术治疗,否则患儿死亡的概率极高。目前虽然临床上有相应的药物治疗可以推迟动脉导管的闭合,但由于其具有较大的副作用,因此亟待开发新的治疗手段维持动脉导管闭合的开放状态,为手术或下一步治疗争取宝贵的时间。然而,对于动脉导管关闭的具体机制目前并没有明确的答案,原因之一是基于人类动脉导管组织,或人类动脉导管平滑肌细胞的研究相对较少。本研究拟通过蛋白质组学实验筛选出人类闭合与开放动脉导管组织中表达明显差异的蛋白质,以及与这些蛋白相关的信号通路,进一步以蛋白质组学的研究结果为基础验证目标蛋白质及信号通路与动脉导管闭合的相关性,并探讨它们维持动脉导管开放状态的具体机制,最终希望了解相应的药物用于临床治疗的可能性。方法:同位素标记的相对和绝对定量的蛋白组学方法(i TRAQ)合并二维液相色谱-串联质谱法(2D LC-MS/MS)来分析人开放及闭合的动脉导管组织中整体的蛋白表达差异情况;Western Blot、q RT-PCR验证蛋白组学实验结果。分离人动脉导管平滑肌细胞在体外培养并使用慢病毒转染的方式敲减MEK1/2,ARHGAP26的表达。使用流式细胞法,细胞活性检测(CCK-8试验),免疫荧光,酶联免疫(Elisa)及小室细胞迁移实验等检测敲减之后动脉导管平滑肌细胞内钙离子浓度,细胞的增殖和迁移能力,细胞外透明质酸(Haluronic acid,HA)的改变情况;Western-blot法检测相关信号通路中关键调控因子表达变化,以期明确丝裂原活化蛋白激酶(MEK1/2)和Rho A/ROCK1信号通路在动脉导管平滑肌细胞增殖和迁移中的作用。使用MEK1/2抑制剂PD0325901对怀孕小鼠进行腹腔注射,墨汁注射法显示新生小鼠动脉导管开放情况。结果:蛋白质组学实验筛选出132种表达差异的蛋白质,在这其中,电压门控钠通道蛋白1.3(SCN3A),肌球蛋白1d(Myo1d),Rho GTP酶激活蛋白26(ARHGAP26)可能与动脉导管的关闭密切相关,MEK1敲减后动脉导管平滑肌细胞内钙离子浓度下降54.1%(P<0.05),MEK2敲减后细胞的迁移能力以及细胞的增殖能力分别降低67.6%(P<0.01)和6.3%(P<0.01),动脉导管平滑肌细胞在体外培养时,当ARHGAP26敲减之后72小时,细胞活性下降46.7%(P=0.02),迁移能力下降50.8%(P=0.004),细胞外透明质酸浓度下降53.6%(P=0.007),PD0325901腹腔注射后,墨汁注射显示新生小鼠在出生后10小时后动脉导管仍未关闭。结论:MEK1/2信号通路与动脉导管的闭合密切相关,MEK1/2抑制剂PD0325901可以有效延长动脉导管闭合的过程;低氧诱导的ARHGAP26缺乏可以通过激活Rho A-ROCK1-PTEN信号通路抑制动脉导管平滑肌细胞的增殖和迁移。更多还原

【Abstract】 Background and Objective:Ductus Arteriosus（DA）-dependent congenital heart disease seriously affects the survival rate of children,especially when the ductus DA is constricted,the infants need surgery in hours,otherwise,the probability of death will be extremely high.At present,although some drugs can delay the closure of the patent ductus arteriosus,due to its severe side effects,there is an urgent need to develop new methods to maintain the patency of DA so as to gain valuable time for surgery or further treatments.However,there is currently no definitive answer to the exact mechanism of DA closure.One of the reasons is that studies based on human DA,or human ductal smooth muscle cells are relatively rare.In this study,proteomics experiments were carried out to screen out significant differences in the expression of proteins in human constricted and patent DA tissues and the signaling pathways associated with these proteins.The results of proteomic studies were further used to validate the target proteins and signaling pathways related with Patent ductus arteriosus,and to discuss their specific mechanisms for maintaining the patency of the DA.Finally,we hope to screen out new drugs for clinical treatment.Methods:The isobaric labeled relative and absolute quantitative-based proteomic methods（i TRAQ）combined with two-dimensional liquid chromatography-tandem mass spectrometry（2D LC-MS/MS）were used to analyze the overall different protein expressions in human patent and constricted human DA tissues;Western Blot,q RT-PCR were completed to validate proteomics experimental results.Isolated human DA smooth muscle cells were cultured in vitro and we knocked down the expression of MEK1/2 and ARHGAP26 through sh MEK1/2 and sh ARHGAP26.Flow cytometry,cell viability assay（CCK-8 assay）,immunofluorescence,Elisa,and cell migration assays were used to detect cytosolic Ca²⁺concentration,cell proliferation,migration and extracellular hyaluronic acid（HA）.Western-blot was performed to detect the expression of regulatory key factors in the signaling pathways in order to clarify the roles of MEK1/2 and Rho A/ROCK1 signaling in the proliferation and migration of DA smooth muscle cells.The pregnant mice were injected intraperitoneally with the MEK1/2 inhibitor PD0325901,and the ink injection results showed the patency of the DA in neonatal mice.Results:132 proteins were screened by proteomic analysis.Among them,voltage-gated sodium channel protein 1.3（SCN3A）,myosin 1d（Myo1d）and Rho GTPase-activating protein 26（ARHGAP26）（P<0.05）turned out tightly related human DA closure.Knockdown of MEK1 reduced the cytosolic Ca²⁺concentration by 54.1%（P<0.05）,MEK2 knockdown reduced the migration and proliferation ability by 67.6%and 6.3%respectively（P<0.01）.72 hours after ARHGAP26 were knockdown in vitro,the cell viability decreased by 46.7%（P=0.02）and the migration ability decreased by 50.8%（P=0.004）.After intraperitoneal injection of PD0325901,ink injections showed that the DAs keep patent even 10 hours after birth in neonatal mice.Conclusion:The MEK1/2 signaling pathway is closely related to the closure of the DA.PD0325901,MEK1/2 inhibitor,can effectively prolong the process of DA closure.Hypoxia-induced ARHGAP26 deficiency can inhibit the proliferation and migration of DA smooth muscle cells through activation of the Rho A-ROCK1-PTEN signaling pathway.更多还原