【作者】 李晓春；

【导师】 陈鹏；

【作者基本信息】 浙江大学 ， 内科学， 2010， 博士

【摘要】 研究背景高血压和动脉粥样硬化(AS)是心血管领域的主要疾病也是其他相关疾病如冠心病、脑中风等的重要致病因素,其病变基础是以血管平滑肌细胞(VSMC)增生和血管内皮细胞(VEC)受损为主要特征。血管平滑肌细胞的增殖和迁移在高血压病的发展和AS的进展中起着重要的作用。AngⅡ作为肾素-血管紧张素系统的重要成分,是VSMC增殖和肥大的主要刺激因子。AngⅡ是许多心血管性疾病的重要促进因素,其中部分原因也是由于其对VSMC和VEC的作用。研究显示有多条细胞信号通路参与了AngⅡ对靶细胞的作用。血红素氧合酶(HO)是血红素分解代谢的起始酶及限速酶,到目前为止被识别的主要有三种,其中HO-1为诱导型。目前的研究提示HO-1在多种细胞的生长、分化等生理和病理过程中起着重要的作用。甲基莲心碱(Nef)是由睡莲科植物莲的成熟种子的绿色胚芽即中药莲心中提取出的一种生物碱,已有研究发现该类生物碱具有抗心律失常、抗血小板聚集、抗氧自由基等作用。然而,对其详细的分子生物学及细胞生物学机制的尚不清楚,从一定程度上限制了甲基莲心碱临床的应用。目前没有关于甲基莲心碱对VSMC和VEC增殖的影响以及对AngⅡ诱导的细胞信号通路干预作用的报道。本实验主要探讨甲基莲心碱对人脐静脉平滑肌细胞(HUVSMC)和人脐静脉内皮细胞(HUVEC)生长的影响以及对其可能的作用机制作进一步的探讨。具体的研究内容：1、甲基莲心碱对HUVSMC和HUVEC增殖的影响；2、甲基莲心碱对AngⅡ诱导的HUVSMC和HUVEC生长的影响；3、分析比较在AngⅡ、甲基莲心碱作用下,细胞内HO-1、ERK1/2蛋白表达的变化。材料与方法1、培养HUVSMC和HUVEC两个细胞系,MTT法检测不同浓度的甲基莲心碱和AngⅡ对HUVSMC和HUVEC细胞增殖的作用。无血清处理细胞24小时后,分别用甲基莲心碱、ZnPP IX、PD98059等预处理细胞1小时后再与AngⅡ共同孵育细胞24小时,分别做各项相关检测。2、流式细胞技术检测各处理因素对HUVSMC和HUVEC细胞周期的作用,流式细胞技术Annexin V FITC法检测甲基莲心碱对HUVSMC细胞凋亡率的影响。3、RT-PCR检测甲基莲心碱和AngⅡ对HUVSMC p21WAF1/CIP1和CyclinD1基因表达水平的作用。4、Western Blot法检测实验各组作用于HUVSMC和HUVEC 24h后细胞HO-1、ERK1／2蛋白表达和ERK1／2磷酸化水平变化,设GAPDH为内参。5、硝酸还原酶法测定HUVEC细胞上清液中NO浓度。6、统计分析：数据均采用SPSS 13.0软件进行统计学处理,结果以均数±SE表示,组间比较用单因素方差分析,多样本均数之间两两比较采用q检验,P<0.05为差异具有显著性意义。结果1、甲基莲心碱抑制AngⅡ诱导的HUVSMC增殖甲基莲心碱能在体外抑制]HUVSMC增殖,且呈浓度时间依赖性。甲基莲心碱不增加HUVSMC细胞凋亡率。甲基莲心碱可有效抑制AngⅡ诱导的HUVSMC的增殖,减少进入S期的细胞数目,使细胞阻滞在G0／G1期。甲基莲心碱(0.5-5.0μmol/L)通过抑制CyclinDl mRNA的表达、促进p21WAF1/CIP1mRNA的表达,使AngⅡ促进的细胞周期进程受阻而拮抗AngⅡ的促HUVSMC增殖作用。2、甲基莲心碱上调HUVSMC的HO-1表达和抑制ERK1／2的磷酸化甲基莲心碱可显著诱导HUVSMC的HO-1蛋白的表达,呈剂量依赖趋势。5μmol/L甲基莲心碱可显著对抗AngⅡ诱导的HO-1表达的减低,同时减弱AngⅡ诱导的ERK1／2磷酸化和细胞增殖。甲基莲心碱的这三种作用均能被HO-1特异性抑制剂ZnPP IX所抑制,此外,ZnPP IX还能进一步促进AngⅡ诱导的ERK1／2磷酸化和细胞增殖。HO-1诱导剂CoPP,则可明显减弱AngⅡ诱导的ERK1／2磷酸化和细胞增殖。ERK阻断剂PD98059可明显减弱AngⅡ对HUVSMC诱导的ERK1／2磷酸化和细胞增殖作用,并阻止ZnPP IX诱导的HUVSMC增殖和显著逆转ZnPP IX诱导ERK1／2磷酸化。3、甲基莲心碱诱导HUVEC增殖及NO的分泌不同浓度(0.1、0.5、1.0、5.0μmol/L)的甲基莲心碱在作用12h、24h、48h后均可诱导HUVEC明显的增生。甲基莲心碱与HUVEC增殖指数的增加呈一定的浓度依赖趋势。ZnPP IX可部分逆转甲基莲心碱的促HUVEC增殖的作用。甲基莲心碱上调HUVEC内HO-1的表达和ERK1／2的磷酸化水平,并明显增加NO的分泌。PD98059和ZnPP IX对甲基莲心碱诱导的ERK1／2磷酸化的上调均有明显的逆转作用,而对HO-1的表达无影响。结论1、甲基莲心碱可在体外抑制HUVSMC的增殖,并通过抑制CyclinDl mRNA的表达,促进P21WAF1/CIP1mRNA的表达而抑制Angll诱导的HUVSMC增殖。2、Angll可抑制体外培养的HUVEC的生长,甲基莲心碱可诱导体外培养的HUVEC的增殖并对抗Angll的生长抑制作用。3、甲基莲心碱可通过上调HO-1的表达抑制]HUVSMC的ERK1／2磷酸化。4、甲基莲心碱可上调HUVEC的HO-1表达和ERK1／2的磷酸化。5、甲基莲心碱可对抗Angll诱导的HUVEC的NO分泌减少。更多还原

【Abstract】 IntroductionProliferation and migration of vascular smooth muscle cells (VSMCs) and endothelial injury play a pivotal role in the development of hypertension and in the progression of atherosclerosis. A vital stimulus for VSMC proliferation and hypertrophy is angiotensinⅡ(AngⅡ), the main effector of the renin-angiotensin system. AngⅡis an important contributing factor to many vascular diseases, in part through its effects on VSMCs and vascular endothelial cells (VECs).Provious studies indicate that many signal molecular, such as mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK)1/2 and P38 have been shown to be implicated in the hypertrophic respense of VSMC to AngⅡ.Heme oxygenases (HOs) are the rate-limiting enzymes in the degradation of heme into carbon monoxide, iron, and biliverdin. To date, three distinct members of the HO-family have been identified. HO-1 is highly inducible by a variety of compounds and different physiologic and pathophysiologic stimuli. Data from current studies suggest that HO-1 plays a vital role in control of cell growth and differentiation.Neferine is a bis-benzylisoquinoline alkaloid extracted from the green seed embryo of Nelumbo nucifera Gaertn, which is effective in preventing the onset of reentrant ventricular tachyarrhythmias. Neferine can inhibit very low density lipoprotein oxidation and platelet aggregation, protect VECs from damage induced by oxygen free radicals. Neferine was shown to exert biological effects on the cardiovascular system consistant with a Calcium channel blocker. However, the precise mechanism of anti-artherosclerosis by which this drug affects the growth of VSMCs and VECs remains obscure. No studies exist that address the effect of neferine on VSMC and VEC proliferation or the interference of neferine with Ang II-induced signaling pathway.Therefore, the aim of this study was to test whether neferine affected the growth of HUVSMCs and HUVECs as well as to identify the underlying signaling pathways in HUVSMCs and HUVECs.To test this assuption, this study was as follows:1) to characterize the effect of neferine on cell proliferation in HUVSMCs and HUVECs; 2) to explore the effect of neferine on cell proliferation induced by AngⅡin HUVSMCs and HUVECs; 3) The role of HO-1 and ERK1/2 signal pathway in the cardioprotective effect of neferine was also investigated.Material and Methods1、The two cell lines:HUVSMC and HUVEC were cultured. Cell proliferation were detected by the MTT assay and flow cytometry analysis. HUVSMCs or HUVECs were respectively pretreated with neferine, ZnPPⅨ, PD98059 for 1h followed by a stimulation with AngⅡfor 24h.2、Cell cycle status of HUVSMC or HUVEC was analyzed with a FACScan flow cytometer by measuring fluorescence from cells stained with propidium iodide.The percentages of cells in the different phases of the cell cycle was analyzed using ModFit 3.0 software. The percentage of apoptosis of HUVSMCs was detected by Annexin V-FITC and PI staining with flow cytometry.3、The gene expressions of p21 WAF1/CIP1 and CyclinDl were detected by RT-PCR at 24h after neferine and or AngⅡin the treated HUVSMC. 4、The phosphorylated and total ERK1/2 proteins and HO-1 protein expression were analyzed by Western Blot at 24h in the treated HUVSMC and HUVEC. For normalization, blots were reprobed with antibodies to detect total amounts of GAPDH.5、NO concentrations in the HUVEC supernatants were detected by using NO enzyme-immunoassay kit based on nitrate reductase method.6、Statistical analysis:Analysed were performed using the SPSS(13.0) statistical software package. Results are expressed as means±SE. Data were analyzed using one-way analysis of variance (ANOVA) followed by the Student Newman-Keuls post hoc Tukey test. Differences between groups were considered to be significant at P<0.05.Result1. Neferine inhibited AngⅡ-induced proliferation Of HUVSMCCompared to the control group, neferine decreased the HUVSMC proliferation in a concentration-dependent manner. Neferine did not increase the percentage of apoptosis of HUVSMC.The proliferation of HUVSMC incubated with AngⅡwas significantly attenuated in the presence of neferine. The antiproliferative effect of neferine was confirmed by cell cycle analysis and the cell percentage of S and G2/M phase decreased. Neferine(0.5-5.0μmol/L) arrested HUVSMCs at the G0/G1 phase through suppressing CyclinDl and promoting p21WAF1/CIP1 mRNA expression.2. Neferine increased HO-1 protein expression and inhibited p-ERKl/2 in HUVSMC stimulated by AngⅡNeferine remarkably induced the expression of HO-1 in a concentration-dependant manner when compared to the control in HUVSMC. Coapplication of neferine(5.0μmol/L) with ZnPP IX significantly inhibited neferine-induced HO-1 expression in cells treated with AngⅡ. The inhibitory effect of neferine on AngⅡ-induced cell proliferation and increased p-ERK1/2 was significantly reversed by ZnPP IX. Moreover, CoPP, a HO-1 inducer, was also capable of preventing AngⅡ-induced proliferation in HUVSMCs, whereas ZnPP IX increased it. Perincubation with PD98059 also decreased of AngⅡ-induced cell proliferation in HUVSMCs. PD98059 inhibited the increase in cell proliferation induced by ZnPP IX.3. Neferine induced HUVEC proliferation and increased the secretion of NO in HUVEC.Compared to the control group, neferine(0.1,0.5,1.0,5.0μmol/L) increased HUVEC proliferation on 12h,24h,48h and promoted the cell cycle in a concentration-dependent manner. Neferine caused the up-regulation of HO-1 expression and increased the secretion of NO in HUVEC. ERK1/2 phosphorylation was significantly increased compared with the control in HUVEC. Coapplication of neferine with ZnPP IX significantly inhibited neferine-induced HO-1 expression and proliferation in HUVEC. PD98059 and ZnPP IX did not affect neferine-induced HO-1 expression,but reversed up-regulated ERK1/2 phosphorylation induced by neferine.Conclusion1、Neferine is capable of inhibiting HUVSMC proliferation and AngⅡ-induced HUVSMC proliferation in vitro. Neferine inhibits the gene expression of CyclinDl mRNA and promotes p21WAF1/CIP1 mRNA expression in AngⅡ-induced HUVSMC.2、AngⅡinhibites the growth of HUVEC in vitro. Neferine promotes HUVEC proliferation and against AngⅡ-induced inhibitory proliferation.3、Neferine up-regulates HO-1 expression and inhibits the phosphorylation of ERK1/2 in HUVSMC.4、Neferine up-regulates HO-1 expression and the phosphorylation of ERK1/2 in HUVEC.5、Neferine can attenuate the decreased the secretion of NO induced by AngⅡin HUVEC.更多还原