【作者】 吴迪；

【导师】 齐国先；

【作者基本信息】 中国医科大学 ， 心血管内科， 2021， 博士

【摘要】 目的:动脉粥样硬化如今已成为危害人类健康的重要疾病,血管内皮受损是其始动因素。血管内皮细胞是是一层有活性的细胞,其完整性及生理特性对人体至关重要。除了动脉粥样硬化过程,血管内皮细胞还参与血管生成、血管收缩与舒张、凝血功能、炎症反应调控、内分泌功能、物质交换等重要的生理和病理过程。血管生成(Angiogenesis)指从毛细血管后微静脉出芽形成新的毛细血管网络,过程极其复杂,其中血管内皮细胞的激活、增殖、迁移、重建形成新的血管和血管网是非常重要的环节,是促进血管生成因子和抑制血管生成因子相互协调作用的结果。正常情况下二者处于平衡状态,一旦平衡打破就会激活血管系统,使血管生成过度或抑制,导致病理性事件。干细胞在血管生成方面的作用一直是研究热点,但是作用机制始终未能完全明确,特别是干细胞通过旁分泌功能对靶细胞的调控作用备受关注。人羊膜来源上皮细胞是干细胞的一种,能够旁分泌大量的生长因子及趋化因子等生物活性因子,旁分泌是其对靶细胞功能进行调控的重要方式之一。本研究的目的是:(1)建立人羊膜来源上皮细胞(hAECs)的体外分离、培养方法,并对其进行表型鉴定和纯度分析。(2)检测人羊膜上皮细胞条件培养液(hAEC-CdM)中的细胞因子,并对其相关信号通路进行富集分析。(3)观察不同浓度的人羊膜上皮细胞条件培养液对体外培养的人主动脉内皮细胞(hAoECs)增殖、迁移及血管生成作用的影响。(4)探究人羊膜上皮细胞条件培养液调节人主动脉内皮细胞相关生理作用的分子机制。研究方法:(1)采用胰蛋白酶消化法分离、体外培养人羊膜上皮细胞,应用免疫荧光染色分析其表型特征。(2)将收集的hAEC-CdM进行液相色谱-串联质谱(LC-MS/MS)检测,并对结果进行GO富集分析和KEGG信号通路分析。(3)实验分组情况:我们设计用ECM培养hAoECs为对照组,标记为Control组;在ECM中加入0.5倍、1倍及2倍浓度的hAEC-CdM设为3个实验组,标记为0.5×CdM组、1×CdM组、2×CdM组,分别用四种培养液培养hAoECs,然后进行相关实验验证hAoECs功能的变化。抑制剂干预实验分组:ECM组(Control)、1×CdM组、抑制剂组(SB431542或K02288)、CdM+抑制剂组。(4)经hAEC-CdM处理24小时、48小时及72小时后,通过CCK-8实验观察不同浓度hAEC-CdM对hAoECs增殖能力的影响。(5)经hAEC-CdM处理12小时及24小时后,通过细胞划痕实验评估不同浓度hAEC-CdM对hAoECs迁移能力的影响。(6)基质胶血管生成实验验证不同浓度hAEC-CdM对hAoECs管腔形成能力的影响。(7)Western Blot检测不同浓度hAEC-CdM对hAoECs TGF-β通路活化的影响。结果:(1)消化下来的人羊膜上皮细胞贴壁后呈多角形,长满后呈现上皮细胞特有的铺路石样,可在体外大量扩增。上皮细胞传至第2代用于细胞鉴定及实验。免疫荧光结果显示:上皮细胞高表达上皮细胞标志物角蛋白CK19,不表达间充质细胞标志物Vimentin,纯度在90%以上。(2)本次质谱分析鉴定蛋白273种,参与信号通路88种。GO富集分析结果显示:人羊膜上皮细胞条件培养液主要调节靶细胞的代谢过程、内吞作用和磷酸化进程;KEGG信号通路分析结果显示:羊膜上皮细胞条件培养液主要影响靶细胞的PI3K-Akt通路、mTOR通路和TGF-β通路。(3)CCK-8实验结果:在24小时节点,不同浓度实验组的hAoECs增殖能力与对照组相比均无显著差别(p>0.05);在48小时节点,0.5×CdM组的hAoECs增殖能力与对照组相比无明显差别(p>0.05),1×CdM组和2×CdM组的hAoECs增殖能力均明显高于对照组(p<0.05);在72小时节点,不同浓度实验组hAoECs增殖能力均明显高于对照组(p<0.05)。根据以上结果可知:hAEC-CdM促进hAoECs增殖,随着hAEC-CdM浓度增加,其对hAoECs增殖的促进作用增强。(4)划痕实验结果:在12小时节点,0.5×CdM组细胞迁移能力与对照组无显著差别(p>0.05),1×CdM组和2×CdM组细胞迁移能力显著高于对照组(p<0.05);在24小时节点,不同浓度实验组细胞迁移能力均明显高于对照组(p<0.05)。根据以上结果可知:hAEC-CdM促进hAoECs迁移,随着hAEC-CdM浓度增加,其对hAoECs迁移的促进作用增强。(5)基质胶血管生成实验结果:对照组hAoECs在6小时开始逐渐形成管腔结构,0.5×CdM组和1×CdM组hAoECs在6小时形成管腔结构多于对照组,且1×CdM组形成的管腔结构多于0.5×CdM组。但是2×CdM组在6小时几乎没有形成管腔结构。根据以上结果可知:低浓度hAEC-CdM促进hAoECs管腔形成。(6)加入不同浓度hAEC-CdM刺激后的Western Blot结果:(1)ALK5-Smad3-Snail-Postn通路相关蛋白的表达:0.5×CdM组、1×CdM组和2×CdM组的ALK5和Smad3蛋白表达水平与对照组相比无显著差异(p>0.05),0.5×CdM组和1×CdM组p-Smad3、Snail和Postn蛋白表达水平明显高于对照组(p<0.05),但2×CdM组p-Smad3、Snail和Postn蛋白表达水平明显低于于对照组(p<0.05)。(2)ALK1-Smad5-Snail-Postn通路相关蛋白的表达:0.5×CdM组、1×CdM组和2×CdM组的ALK1和Smad5蛋白表达水平与对照组相比无显著差异(p>0.05),0.5×CdM组和1×CdM组p-Smad5、Snail和Postn蛋白表达水平明显高于对照组(p<0.05),但2×CdM组p-Smad5、Snail和Postn蛋白表达水平明显低于于对照组(p<0.05)。(7)加入抑制剂后的Western Blot结果:(1)加入SB431542后p-Smad3及其下游相关蛋白的western blot结果:加入抑制剂SB431542后,相较于对照组Smad3的蛋白表达水平无显著差异(p>0.05),p-Smad3、Snail和Postn的蛋白表达水平显著降低(p<0.05)。1×CdM组p-Smad3、Snail和Postn的表达水平相较于对照组显著升高(p<0.05),说明1×CdM促进了上述蛋白的表达;加入SB431542后,(CdM+SB)组p-Smad3、Snail和Postn的蛋白表达水平显著降低(p<0.05),说明SB431542一定程度上抑制了1×CdM对hAoECs上述蛋白表达的促进作用。(2)加入K02288后p-Smad5及其下游相关蛋白的western blot结果:加入抑制剂K02288后,相较于对照组Smad5的蛋白表达水平无显著差异(p>0.05),p-Smad5、Snail和Postn的蛋白表达水平显著降低(p<0.05)。1×CdM组p-Smad5、Snail和Postn的表达水平相较于对照组显著升高(p<0.05),说明1×CdM促进了hAoECs的上述蛋白的表达;在加入K02288后(CdM+K02288组)p-Smad5、Snail和Postn的蛋白表达水平显著降低(p<0.05),说明K02288在一定程度上抑制了1×CdM对hAoECs上述蛋白表达的促进作用。(8)加入抑制剂后的血管生成实验结果:(1)使用1×CdM预培养主动脉内皮细胞24小时后进行基质胶血管生成实验,对照组细胞在6小时开始逐渐形成管腔结构,1×CdM组细胞形成的管腔结构明显多于对照组。(2)加入SB431542抑制Smad3磷酸化后,在6小时节点,SB431542组未见管腔结构形成,而(CdM+SB431542)组形成管腔数目明显少于1×CdM组,说明抑制Smad3磷酸化水平确实可以抑制hAEC-CdM促hAoECs血管生成的能力。(3)加入K02288抑制Smad5磷酸化后,在6小时节点,K02288组未见管腔结构形成,而(CdM+K02288)组形成管腔数目明显少于1×CdM组,说明抑制Smad5磷酸化水平确实可以抑制hAEC-CdM促hAoECs血管生成的能力。结论:(1)成功分离培养人羊膜上皮细胞,高表达CK19,不表达Vimentin,纯度在90%以上,在体外培养条件下快速增殖。(2)本次质谱分析鉴定蛋白273种,参与信号通路88种。GO富集分析结果提示人羊膜上皮细胞条件培养液主要调节靶细胞的代谢过程、内吞作用和磷酸化进程;KEGG信号通路分析结果提示人羊膜上皮细胞条件培养液主要影响靶细胞的PI3K-Akt通路、mTOR通路和TGF-β通路。(3)hAEC-CdM促进hAoECs增殖及迁移。(4)低浓度hAEC-CdM激活TGF-β信号通路促进hAoECs管腔形成的能力,而高浓度hAEC-CdM的抑制作用可能与其抑制hAoECs的Snail和Postn表达相关。更多还原

【Abstract】 Objective: Atherosclerosis is a severe thereat to health and often starts with endothelium injury.Endothelial cells are a layer of active cells,whose integrity and biological characteristics are of great importance.Besides Atherosclerosis,endothelial cells participate in a lot of significant biological and pathological processes,such as angiogenesis,contraction and dilation of blood vessels,coagulation,inflammation and many more.Angiogenesis refers to the process of sprouting from existing vessels as well as forming new blood vessel networks,which is extremely complicated.This process is dependent on the balance between pro-angiogenic and anti-angiogenic factors.Once the balance is broken,the vascular system will be activated,causing overactivation or inhibition of angiogenesis which leads to pathological events.The role of stem cells in angiogenesis has always been a research hotspot,but the mechanism has not been fully understood,particularly related to regulating target cells by stem cells through paracrine function.Human amniotic epithelial cells(hAECs),as one kind of stem cells,can paracrine a large number of biological active factors such as growth factors and chemokines,which is an important means of regulating the function of target cells.The purposes of our study :(1)In vitro isolate hAECs from human amnion membranes,and perform phenotypic identification and purity analysis.(2)Detect the cytokines in the conditioned midium of hAECs(hAEC-CdM),and perform enrichment analysis of the related signaling pathways.(3)Investigate the biological potential and behavior of hAEC-CdM of different concentrations on proliferation,migration and angiogenesis of cultured human aortic endothelial cells(hAoECs).(4)Investigate the related molecular mechanism of the conditioned midium(CdM)of hAECs on human aortic endothelial cells.Methods:(1)Human amniotic epithelial cells(hAECs)were isolated from amnion membranes by 0.25% trypsin and analyzed for their phenotypic characteristics by immunofluorescence staining.(2)LC MS/MS was performed to test the proteins in the collected hAEC-CdM,and GO Analysis and KEGG Pathway Analysis were performed to analyse the LC MS/MS results.(3)We designed ECM group as control,and ECM with different concentrations of hAEC-CdM as test groups,marked as Control group,0.5×CdM group,1×CdM group and 2×CdM group.For inhibitor assays we set 4 groups:ECM group(Control),1×CdM group,inhibitor group(SB431542 or K02288)and CdM+inhibitor group.(4)We compared the effects of different concentrations of hAEC-CdM on hAoECs proliferation by means of CCK-8 assays after stimulated by hAEC-CdM for 24,48 and 72 hours.(5)We compared the effects of different concentrations of hAEC-CdM on hAoECs migration by means of scratch assays after stimulated by hAEC-CdM for 12 and 24 hours.(6)We compared the effects of different concentrations of hAEC-CdM on hAoECs angiogenesis by means of matrigel assays after stimulated by hAEC-CdM for 24 h.(7)After stimulated by different concentrations of hAEC-CdM,we performed western blot to test the protein expression levels of activated TGF-β signaling pathway of hAoECs.Results:(1)The isolated hAECs are of polygon shape,and after proliferation they turned into paving stone shape.We used passage 2 for identification and the assays.Immunofluorescence results showed that hAECs highly expressed CK19 and did not express vimentin,and the purity was above 90%.(2)Our LC MS/MS results identified273 proteins in hAEC-CdM and 88 signaling pathways were involved.GO analysis showed that hAEC-CdM mainly regulated the metabolic process,endocytosis process and phosphorylation process of target cells.KEGG pathway analysis showed that hAEC-CdM mainly affected the PI3K-Akt pathway,mTOR pathway and TGF-β pathway of target cells.(3)CCK-8 assays: After stimulated by hAEC-CdM for 24 hours,there was no difference of hAoECs proliferative ability between test groups and control group(p>0.05).After stimulated by hAEC-CdM for 48 hours,there was no difference of hAoECs proliferative ability between 0.5×CdM group and control group(p>0.05),while1×CdM group and 2×CdM group both showed better proliferative ability than control group(p<0.05).After stimulated by hAEC-CdM for 72 hours,hAoECs of each test group showed better proliferative ability than control group(p<0.05).The higher concentration of hAEC-CdM,the better proliferative ability of hAoECs.(4)Scratch assays: After stimulated by hAEC-CdM for 12 hours,there was no difference of hAoECs migration ability between 0.5×CdM group and control group(p>0.05),while 1×CdM group and2×CdM group both showed better migration ability than control group(p<0.05).After stimulated by hAEC-CdM for 24 hours,hAoECs of each test group showed better migration ability than control group(p<0.05).So the higher concentration of hAEC-CdM,the better migration ability of hAoECs.(5)Matrigel assays: After stimulated by lyophilized powder solution for 24 hours,we performed matrigel assays to evaluate the angiogenesis ability of hAoECs.At 6 hour point,control group gradually had tube formation.Both 0.5×CdM group and 1×CdM group showed more tube formation than control group,and there was more tube formation of 1×CdM group than 0.5×CdM group,which means low concentraion of hAEC-CdM promoted hAoECs tube formation.(6)Western blots after stimulated by different concentrations of hAEC-CdM:(1)ALK5-Smad3-Snail-Postn signaling pathway: There was no significant difference of ALK5 and Smad3 expression between 0.5×CdM group,1×CdM group,2×CdM group and control group(p>0.05).Expression levels of p-Smad3,Snail and Postn of 0.5×CdM group and 1×CdM group were significantly higher than control group(p<0.05),while that of 2×CdM group were significantly lower than control group(p<0.05).(2)ALK1-Smad5-Snail-Postn signaling pathway: There was no significant difference of ALK1 and Smad5 expression of 0.5×CdM group,1×CdM group,2×CdM group and control group(p>0.05).Expression levels of p-Smad5,Snail and Postn of 0.5×CdM group and 1×CdM group were significantly higher than control group(p<0.05),while that of 2×CdM group were significantly lower than control group(p<0.05).(7)Western blots after interfered by inhibitors:(1)Western blots of p-Smad3 and its downstream genes after interfered by SB431542: After interfered by SB431542,there was no significant difference of Smad3 expression between SB431542 group and control group(p>0.05),while the expression levels of p-Smad3,Snail and Postn were significant lower than control group(p<0.05).p-Smad3,Snail and Postn expression levels of 1×CdM group were significantly higher than control group(p<0.05),which means 1×CdM promoted the above proteins expression.After disturbed by SB431542,the expression levels of p-Smad3,Snail and Postn of(CdM+SB)were significantly lower than 1×CdM group(p<0.05),which means SB431542 inhibited the promotion of 1×CdM on the above protein expression.(2)Western blots of p-Smad5 and its downstream genes after interfered by K02288: After interfered by K02288,there was no significant difference of Smad5 expression between K02288 group and control group(p>0.05),while the expression levels of p-Smad5,Snail and Postn were significant lower than control group(p<0.05).p-Smad5,Snail and Postn expression levels of 1×CdM group were significantly higher than control group(p<0.05),which means 1×CdM promoted the above proteins expression.After disturbed by K02288,the expression levels of p-Smad5,Snail and Postn of(CdM+SB)were significantly lower than 1×CdM group(p<0.05),which means K02288 inhibited the promotion of 1×CdM on the above protein expression.(8)After applying inhibitors:(1)After cultivating hAoECs by 1×CdM for 24 h,we applied matrigel assays.Control group started to have tube formation at 6h point,and 1×CdM group had more tube formation than control group.(2)After applying SB431542 inhibiting Smad3 phsophorylation,at 6h point,SB431542 group had no tube formation,while CdM+SB431542 group had much less tube formation than 1×CdM group,which means inhibition of Smad3 phsophorylation could inhibite the promotion of hAEC-CdM on hAoECs angiogenesis.(3)After applying K02288 inhibiting Smad5 phsophorylation,at6 h point,K02288 group had no tube formation,while CdM+K02288 group had much less tube formation than 1×CdM group,which means inhibition of Smad5 phsophorylation could inhibite the promotion of hAEC-CdM on hAoECs angiogenesis.Conclusion:(1)hAECs were successfully isolated and cultured in vitro,with high expression of CK19 and no expression of vimentin,purity of more than 90%.hAECs could rapidly proliferate in vitro culture conditions.(2)Our LC MS/MS results identified273 proteins in hAEC-CdM and 88 signaling pathways were involved.GO analysis showed that hAEC-CdM mainly regulated the metabolic process,endocytosis process and phosphorylation process of target cells.KEGG pathway analysis showed that hAEC-CdM mainly affected the PI3K-Akt pathway,mTOR pathway and TGF-β pathway of target cells.(3)hAEC-CdM promoted hAoECs proliferation and migration.(4)Low concentrations of hAEC-CdM promoted hAoECs angiogenesis by activating its TGF-βsignaling pathway.High concentration of hAEC-CdM might inhibit hAoECs angiogenesis by inhibiting the downstream genes Postn and Snail expression of TGF-βsignaling pathway of hAoECs.更多还原