【作者】 吴德华；

【导师】 姜桢； 缪长虹； 陈瑞珍；

【作者基本信息】 复旦大学 ， 麻醉学， 2008， 博士

【摘要】 第一部分:血管紧张素Ⅱ对肺微血管内皮细胞Apelin-APJ mRNA表达的影响目的研究血管紧张素Ⅱ（AngⅡ）对肺微血管内皮细胞Apelin-APJ mRNA表达的影响。方法培养新生鼠肺微血管内皮细胞（PMVECs）,利用RT-PCR检测PMVECsApelin-APJmRNA表达。取2-4代细胞,以5×10⁵个/孔密度接种至6孔板,培养至80%融合后,进行药物干预实验:①加AngⅡ入6孔板中,至终浓度分别为0、10^-9、10^-8、10^-7、10^-6M（mol/L）,每组设立4个复孔,继续培养24h终止实验,检测不同浓度AngⅡ对PMVECs Apelin-APJmRNA表达的影响;②加AngⅡ入6孔板至终浓度为10^-7M,分别在0h、1h、6h、12h、24h、48h时间点终止实验。同时在0h、24h、48h点设立空白对照,加入等体积PBS。每组4个复孔。检测不同时间点10^-7M AngⅡ对PMVECs Apelin-APJmRNA表达。结果①PMVECs存在Apelin-APJmRNA表达。②与对照组（OM AngⅡ）比较,10^-9M AngⅡ作用24h后Apelin mRNA表达上调（P＜0.01）,随着AngⅡ浓度上升,Apelin mRNA表达呈浓度依赖性持续下调（P＜0.05或P＜0.01）;不同浓度AngⅡ(10^-9-10^-6M)呈浓度依赖性导致APJmRNA表达下降（P＜0.05或P＜0.01）。③10^-7MAngⅡ作用于PMVECs后,Apelin mRNA表达在6h内增加（P＜0.05）,其中在1h达到高峰（P＜0.01）,6h后Apelin mRNA表达随时间延长明显降低（P＜0.01）;APJ mRNA表达在0h-12h内有增加趋势,12h后APJ mRNA表达随10^-7M AngⅡ作用时间延长明显下调（P＜0.01）;0h、24h、48h时间点空白对照Apelin-APJmRNA表达无明显改变（P＞0.05）。结论AngⅡ对PMVECs Apelin-APJ基因表达水平有明显影响,Apelin-APJ系统可能参与AngⅡ对内皮细胞损伤机制。第二部分:Apelin对肺血管内皮细胞增殖和凋亡的影响目的探讨Apelin对肺血管内皮细胞增殖与凋亡的影响。方法培养新生大鼠肺微血管内皮细胞（PMVECs）,取2-4代PMVECs用于实验:①以2000个/孔的密度接种至96孔板,分为3组:对照组加入等容量PBS;低浓度组(10^-8M Apelin组)加入10^-6M Apelin;高浓度组(10^-6M Apelin组)加入10^-6MApelin。每组6个复孔。48h后用四甲基偶氮唑蓝比色法（MTT法）检测各组OD值,计算细胞增殖率。②以5×10⁵传代至25ml的培养瓶,分为5组:对照组加入等容量PBS;低浓度组(10^-8M Apelin组)加入10^-8M Apelin;高浓度组(10^-6M Apelin组)加入10^-6M Apelin;AngⅡ组加入10^-7M AngⅡ;AngⅡ+Apelin组加入10^-8MApelin,10min后加入10^-7M AngⅡ。每组5个复孔。24h后用流式细胞仪检测细胞凋亡率。结果①与对照组比较,低浓度Apelin和高浓度Apelin都可导致PMVECs OD值增加,增殖率增加2.5倍以上（P＜0.01）。低浓度组和高浓度组之间没有统计学差异。②与对照组比较,低浓度Apelin组和高浓度Apelin组的PMVECs凋亡率降低50%以上（P＜0.05）,AngⅡ组PMVECs凋亡率明显升高（P＜0.01）;与AngⅡ组比较,AngⅡ+Apelin组PMVECs凋亡率明显降低（P＜0.01）。结论Apelin促进体外培养大鼠PMVECs增殖,抑制PMVECs凋亡。第三部分:Apelin对肺微血管内皮细胞保护作用及其释放NO的机制研究目的探讨Apelin对血管紧张素Ⅱ（AngⅡ）诱导肺血管内皮细胞释放ET-1的影响和刺激一氧化氮（NO）的释放机制。方法培养新生鼠肺微血管内皮细胞（PMVECs）,取2-4代PMVECs用于实验。①Apelin在不同时间点对PMVECs释放NO的影响:以1×10⁵个/孔密度接种至24孔板,80%融合后,无血清培养24h,分为2组:Apelin组,加入Apelin至终浓度为10^-8M;对照组,加入等量无血清DMEM培养液。每组设立4个复孔。继续培养,并分别在0min、2min、5min、15min、30min五个时间点取培养液测NO浓度。②Apelin对AngⅡ诱导PMVECs释放ET-1的影响:分为4组:AngⅡ组,加入AngⅡ至终浓度为10^-7M;AngⅡ+10^-8M Apelin组,加入10^-7M AngⅡ前5min加入10^-8M Apelin预处理;AngⅡ+10^-6M Apelin组,加入10^-7M AngⅡ前5min加入10^-6M Apelin预处理;对照组,加入等量无血清DMEM培液。每组设立4个复孔。继续培养,并分别在0min、30min、6h、24h四个时间点取培养液检测ET-1浓度。③Apelin对PMVECs Akt/eNOS磷酸化影响:以5×10⁵个/孔密度接种至6孔板,80%融合后,无血清培养24h,分为3组:Apelin组,加入Apelin至终浓度为10^-8M;Apelin+Akt inhibitor组,加入Apein前用5uM浓度的Akt抑制剂预处理30min;对照组,加入等量DMEM培液。每组设立3复孔。继续培养5min后立即提取总蛋白,用于Western Bolt检测磷酸化Akt和磷酸化eNOS表达。结果①Apelin在不同时间点对PMVECs释放NO的影响:在PMVECs培养液中加入10^-8M的Apelin后,与对照组比较,培养液中NO浓度在2min、5min、15min时间点都出现增加（P＜0.01或P＜0.05）;其中在5min时间点达到高峰（与对照组比较,P＜0.01）;在30min时间点,NO浓度与对照组比较无统计学差异。②Apelin对AngⅡ诱导PMVECs释放ET-1的影响:与对照组比较,AngⅡ组培养液中30min、6h、24h时间点的ET-1浓度都明显升高（P＜0.05）。与AngⅡ组比较,AngⅡ+10^-8M Apelin组培养液中30min、6h、24h时间点的ET-1浓度都明显降低（P＜0.05）,接近对照组。与AngⅡ组比较,AngⅡ+10^-8 Apelin组的ET-1浓度有降低趋势,但无统计学意义。③Apelin对PMVECs Akt/eNOS磷酸化影响:与对照组比较,Apelin组PMVECs Akt和eNOS磷酸化表达明显增加（P＜0.01）。与Apelin组比较,Apelin+Akt inhibitor组PMVECs eNOS磷酸化表达明显降低（P＜0.01）。结论Apelin能促进PMVECs释放NO,抑制AngⅡ诱导的ET-1释放。Akt/eNOS磷酸化增加是Apelin促进NO释放机制之一,并可能参与细胞保护。更多还原

【Abstract】 PartⅠ:The effect of angiotensinⅡon expression of Apelin-APJ mRNA in pulmonary microvascular endothelial cellsObjective Exploring the effect of angiotensinⅡ（AngⅡ） on expression of Apelin-APJ mRNA in pulmonary microvascular endothelial cells（PMVECs）.Methods Pulmonary microvascular endothelial cells from neonatal rat of 24h after birth were cultured.Expressions of Apelin-APJ mRNA in endothelial cells were determined by reverse transcription-polymerase chain reaction（RT-PCR）.PMVECs were used between passages 2 and 4.5×10⁵ cells grown in 6-well plates at subconfluence were stimulated with various concentrations of AngⅡ(0、10^-9、10^-8、10^-7、10^-6M) for analyzing apelin and APJ mRNA expressions by RT-PCR at 24hr and with AngⅡof 10^-7 M for apelin and APJ mRNA expression at different time points of 0hr,1hr,6hr, 12hr,24hr,48hr.At the same time analyzing their expressions in blank groups at three time points of 0hr,24hr and 48hr without stimulation of AngⅡ.every experience was repeated for 4 times.Results①PMVECs expressed apelin and APJ mRNA determined by RT-PCR.②compared with control group（OM AngⅡ）,10^-9 M AngⅡinduced upregulation of apelin mRNA expressions in PMVECs at 24hr（P＜0.01）, higher concentrations of AngⅡ(10^-8、10^-7、10^-6M) induced concentrationdependent downregulation of apelin mRNA expressions（P＜0.05 or P＜0.01）; different concentrations of AngⅡ(10^-9、10^-8、10^-7、10^-6M) concentrationdependently downregulated expressions of APJ mRNA.③10^-7 M AngⅡupregulated apelin mRNA expressions in short time（within 6hr,P＜0.05） with a peak at 1hr（P＜0.01）,and markedly downregulated them 6 hrs later （P＜0.01）;10^-7M AnglI also markedly time-dependently downregulated APJ mRNA expressions after 12 hrs（P＜0.01）;apelin and APJ mRNA expressions did not significantly change in PMVECs without treatment with AngⅡat 3 time points of 0hr,24hr and 48hr.Conclusion AngⅡcan markedly effect on apelin and APJ mRNA expressions in PMVECs,and apelin-APJ system may involve in the mechanism of injury of AngⅡon endothelial cells.PartⅡ:Effects of Apelin on proliferation and apoptosis in pulmonary vascular endothelial cellsobjective To investigate the effects of apelin on proliferation and apoptosis in pulmonary vascular endothelial cells.Methods Pulmonary microvascular endothelial cells（PMVECs） between passage 2 and 4 cultured from neonatal rat of 24 hours after birth were starved serum-free for 24hours,and then were used to following studies:①2×10³ cells per well grown in 96-well plate were divided randomly into 3 groups:Control group（C group） was added with equal volume of PBS;Lower concentration group(10^-8 M group) with 10^-8 M of apelin; Higher concentration group(10^-6 M group) with 10^-6 M of apelin.MTT assay was used to assess cell proliferation rates after cells were continuously incubated for 48 hours.②5×10⁵ cells grown in cultured-bottles of 25 ml were divided into 5 groups:Control group were added with equal volume of PBS;Lower concentration group(10^-8 M group) with 10^-8 M of apelin; Higher concentration group(10^-6 M group) with 10^-6 M of apelin; AngiotensinⅡ（AngⅡ） group with 10^-7 M of AngⅡ;AngⅡ+Apelin group with 10^-7 M of AngⅡand 10^-8M of apelin.The apoptosis rates of PMVECs in each group were detected by flow cytometry after the cells were incubated for 24 hours.Results①The proliferation rates of the PMVECs significantly increased in both lower and higher concentration of apelin groups compared to control group（P＜0.01）,and there were not significantly different between lower and higher concentration of apelin groups.②The apoptosis rates of the PMVECs significantly decreased in both lower and higher concentration groups but dramatically increased in AngⅡgroup,compared to control group（P＜0.05 or P＜0.01）;The apoptosis rates were lower in AngⅡ+Apelin group than in AngⅡgroup（P＜0.01）.Conclusion Apelin can promote proliferation of PMVECs and inhibit apoptosis.PartⅢ:Protective effects of Apelin on pulmonary microvascular endothelial cells and the mechanism of it’ s promoting NO releaseObjective To investigate the effects of Apelin on release of ET-1 induced by AngiontensinⅡand it’ s mechanism of stimulating NO release in pulmonary microvascular endothelial cells（PMVECs）.Methods PMVECs between passage 2 and 4 cultured from neonatal rat of 24 hours after birth were starved serum-free for 24 hours,and then used to following studies.①The effects of Apelin on release of NO in PMVECs at different times: 1×10⁵ cells per well grown in 24-well plate were divided randomly into 2 groups:Apelin group was added with 10^-8 M Apelin;control goup with equal volume of serum-free DMEM.Then concentration of NO in cultural medium were detected in both groups at five different time points of 0min,2min,5min,15min and 30min.②The effects of Apelin on ET-1 release in PMVECs induced by AngiotensinⅡ:PMVECs grown in 24-well plats were randomly divided into 4 groups: AngⅡgroup was added with 10^-7M Apelin;AngⅡ+10^-8M Apelin group incubated with 10^-8M Apelin at 5 min prior to stimulation of 10^-7M AngⅡ; AngⅡ+10^-6 M Apelin group with 10^-6M Apelin 5min prior to 10^-7M AngⅡ; control group with equal volume of serum-free DMEM.ET-1 concentrations in cultural medium were measured in all groups at different time points of 30min,6hr and 24hr.③The effects of Apelin on phosphorylations of endothelial nitric-oxide synthase and Akt:5×10⁵ cells per well grown in 6-well plates were divided into 3 groups:Apelin group was added with 10^-8M of Apelin; Apelin+Akt inhibitor group was pretreated with 5uM of Akt inhibitor 30min prior to incubated with Apelin;control group with equal volume of serum-free DMEM.5min later western blot was used to assay protein expressions of phosphorylations of endothelial nitric-oxide synthase and Akt.Results①The effects of Apelin on release of NO in PMVECs at different times: compared with control group,concentrations of NO in cultural medium increased at the three time points of 2min,5min,and 15min（P＜0.01 or P＜0.05）,with the peak at the point of 5min（P＜0.01）;but concentration of NO did not changed statistically at the point of 30min compared to control group.②The effects of Apelin on ET-1 release in PMVECs induced by AngiotensinⅡ:1) Compared with control group,concentrations of ET-1 in cultural medium of AngⅡgroup at three points of 30min,6hr and 24hr increased significantly（P＜0.05）.2) Compared with AngⅡgroup,concentratins of ET-1 in cultural medium of AngⅡ+10^-8 Apelin group at the three points decreased significantly（P＜0.05）,and close to control group.3) Compared with AngⅡgroup,concentratins of ET-1 in cultural medium of AngⅡ+10^-6 Apelin group trended to decrease but without statistical significance.③The effects of Apelin on phosphorylations of endothelial nitric-oxide synthase and Akt:1) Compared with control group,Apelin induced increase of protein expressions of phosphorylation of both Akt and eNOS significantly in Apelin group（P＜0.01）.2) Compared with Apelin group,protein expressions of phosphorylation of eNOS significantly decreased（P＜0.01）.Conclusion Apelin can induced NO release and attenuated ET-1 release induced by AngⅡin PMEVCs.Promoting phosphorylation of Akt/eNOS was one of mechanisms to induce NO release and may be involved in protective effect on PMVECs for Apelin.更多还原