内皮抑素对人脐静脉内皮细胞(HUVEC) 及体外微血管 模型的作用及其可能的机制

目的：探讨内皮抑素对人脐静脉内皮细胞（HUVEC）及体外微血管模型的作用及其可能的机制。方法：1．MTT法检测不同浓度（10～50μg／ml）内皮抑素作用72h和30μg／ml内皮抑素作用不同时间（24～72h）对HUVEC细胞的影响;2、电镜观察HUVEC细胞超微结构的变化;3．光镜下观察内皮抑素（30μg／ml）对体外人造血管模型的影响。结果：1．MTT检测显示,内皮抑素（20～50μg／ml）能抑制HUVEC细胞的增殖（P〈0．05,P〈0．01）,具有剂量-时间依赖性。2．电镜观察,HUVEC细胞内皮抑素作用组均出现凋亡改变。3．光镜观察,内皮抑素能抑制新生血管的形成,并能破坏新生的血管网。结论：内皮抑素能抑制人脐静脉血管内皮细胞HUVEC的增殖,并具有时间一剂量依赖性,机制可能为诱导细胞凋亡。提示,内皮抑素可能通过诱导HUVEC的凋亡抑制其增殖,并能破坏新生的血管。内皮抑素可能以此抑制机体肿瘤的生长与转移。     

hESC衍生间充质干细胞可显著促进体外血管生成

人胚胎干细胞(hESC)是具有体外无限增殖能力和多向分化潜能的一类亚全能干细胞,在特定的条件下可被诱导分化为机体的各种细胞包括间充质干细胞(MSC)。该研究以人脂肪间充质干细胞(ADSC)和脐带间充质干细胞(UC-MSC)为对照,对hESC衍生间充质干细胞(hESC-MSC)在体外血管生成中的作用进行了系统的研究,包括其条件培养上清对脐静脉血管内皮细胞(HUVEC)的功能和向血管内皮细胞分化潜能的影响。研究发现,hESC-MSC的条件培养上清可显著促进HUVEC的增殖和迁移,其促进HUVEC增殖的作用显著高于UC-MSC的条件培养上清;hESC-MSC经不同血管内皮细胞诱导方案诱导后均具有体外类血管网络生成的能力,其网络长度均显著高于ADSC和UC-MSC经单一血管内皮细胞诱导方案诱导后的长度。因此,hESC-MSC可显著促进体外血管生成,提示该细胞有望成为一种有效的治疗新型心血管疾病的细胞。     

人内皮抑素抗肿瘤相关肽基因的克隆表达及活性研究

目的 克隆并表达人内皮抑素抗肿瘤相关肽,检测其生物活性。方法 人工合成人内皮抑素1—30位氨基酸（30肽,序列25—31由RGIRGAD改为RGDRGD）所对应的核苷酸序列,连接到质粒pTYB2中,再转化至大肠埃希菌BL21（DE3）中表达,几丁质亲和层析树脂一步纯化30肽。通过MTT法、鸡胚绒毛尿囊膜（CAM）实验、小鼠体内抑瘤实验比较30肽和内皮抑素抗肿瘤活性。结果 MTT证实30肽体外对人脐静脉内皮细胞（HUVEC）、胃癌7901细胞（SGC-7901）半数抑制浓度IC50为36μg／ml、47μg／nl,显著低于内皮抑素IC50179μg／ml、202μg／ml。CAM实验中30肽对血管的抑制作用更强。30肽在小鼠体内抑瘤率47．8％,效果优于内皮抑素28．7％。结论 30肽具有更强抗肿瘤活性,有可能成为治疗肿瘤的一种新药物。     

外源性端粒酶基因对人脐静脉内皮细胞的影响

为了观察外源性端粒酶逆转录酶基因(hTERT)在人脐静脉血管内皮细胞(HUVEC)的表达及对细胞功能和生长的影响。采用逆转录病毒载体转导的方法,将hTERT基因转入HUVEC,检测基因转导后内皮细胞端粒酶的活性和生物学特性。结果发现hTERT转导后细胞端粒酶表达阳性,未转导的亲代细胞为阴性;转导细胞的体外生存时间延长但未永生化,而内皮细胞黏附分子表达的功能未受影响。     

HAT抑制剂诱导内皮细胞凋亡并抑制体外成管

乙酰化(acetylation)修饰是具有重要生物学意义的蛋白质翻译后修饰方式,由相互拮抗的组蛋白乙酰转移酶(histone acetyltransferase,HAT)和组蛋白去乙酰化酶(histone deacetylase,HDAC)所催化。近期发现HDAC抑制剂可通过抑制内皮细胞增殖等方式调节血管新生(angiogenesis),但HAT抑制剂是否有相反效应尚不明确。本研究观察了HAT抑制剂Garcinol对体外培养的人脐静脉内皮细胞HUVEC增殖、凋亡、迁移及成管的影响。结果发现,Garcinol在2.5~20μmol/L的范围内可剂量依赖性减少HUVEC的活细胞数目。Garcinol处理对HUVEC的细胞周期无明显影响,但Hochest染色、TUNEL染色及流式细胞术均发现Garcinol处理可显著诱导HUVEC的凋亡。此外,Garcinol处理还可抑制HUVEC的迁移和体外成管。以上结果提示:HAT抑制剂可能通过诱导内皮细胞凋亡而抑制血管新生,这可能是其抗肿瘤效应的新机制。     

改良内皮抑素对人脐静脉内皮细胞抑制作用的实验研究

目的:探讨改良内皮抑素(RGDRGD-ES)对人脐静脉内皮细胞(HUVEC)的抑制作用,摸索RGDRGD-ES对HUVEC细胞抑制作用的相对最佳作用浓度和时间。方法:通过快速定点诱变PCR方法获得含有RGDRGD膜序的改良人内皮抑素基因,并构建其原核表达载体。表达、纯化改良内皮抑素(RGDRGD-ES),运用MTT法和流式细胞仪检测RGDRGD-ES对人脐静脉内皮细胞的抑制作用。结果:1.诱变了ES基因,获得了改良的RGDRGD-ES基因,并成功构建其原核表达载体。2.获得了RGDRGD-ES蛋白。3.改良的RGDRGD-ES能够有效抑制人脐静脉内皮细胞的生长(P<0.01);抑制率随着药物浓度(10μg/ml、20μg/ml、30μg/ml)的增加和作用时间(24 h、48 h、72 h)的延长而逐渐增加,具有浓度和时间依赖性(P<0.01);而30μg/ml与40μg/ml、50μg/ml组间、72 h与96 h组间无明显差异(P>0.05)。4.细胞凋亡率(作用24 h)具有药物浓度(10μg/ml、20μg/ml、30μg/ml)依赖性(P<0.01),30μg/ml与40μg/ml、50μg/ml组间凋亡率无明显差异(P>0.05)。结论:成功构建了改良RGDRGD-ES基因的原核表达载体,RGDRGD-ES蛋白在30μg/ml浓度作用72小时条件下能够有效抑制人脐静脉内皮细胞的生长,改良内皮抑素(RGDRGD-ES)对HUVEC的抑制作用较ES明显提高。     

腺相关病毒载体介导的Endostatin及K1-5蛋白抗肿瘤血管生成协同作用

为研究腺相关病毒载体介导的内皮抑素(Endostatin)及K1-5蛋白抗肿瘤血管生成协同作用,实验中用含有Endostatin及K1-5基因的重组病毒rAAV-hE和rAAV-K5分别感染BHK-21细胞,并表达分泌出内皮抑素和K1-5蛋白,EIA法测定培养液上清中内皮抑素浓度达36.42ng/m l,免疫斑点印迹实验表明rAAV-K5可介导K1-5的体外表达。重组腺相关病毒(rAAV)介导所表达的内皮抑素和K1-5蛋白对血管内皮细胞增殖具有抑制作用。二者对人脐静脉内皮细胞(ECV-304)的抑制率分别为68.1%和63.1%,对牛毛细血管内皮细胞(BCE)的抑制率分别为41.6%和34.0%。同时二者还有协同效应,用rAAV-hE和rAAV-K5同时感染BHK-21细胞,表达产物对人脐静脉内皮细胞和牛毛细血管内皮细胞的抑制率分别为70.8%和43.8%。动物实验表明,重组病毒rAAV-hE和rAAV-K5转入荷有人肺肿瘤细胞的裸鼠,对肿瘤细胞生长具有抑制作用,同时二者具有协同抗肿瘤作用。     

桂皮醛上调Nrf2改善衰老大鼠血管内皮功能障碍

目的:探讨桂皮醛对衰老大鼠模型血管内皮功能的影响及相关机制。方法:通过建立24月龄自然衰老的Sprague Dawley(SD)大鼠模型及第14代的衰老人脐静脉内皮细胞模型(HUVECs),以桂皮醛(10μM)进行体外干预,分别以DHE染色和DAF-2DA染色观察桂皮醛对颈动脉内膜和HUVEC中超氧阴离子、一氧化氮水平的影响。以微血管张力测定仪观察桂皮醛对乙酰胆碱诱导的颈动脉内皮依赖性舒张功能和硝酸甘油诱导的非内皮依赖性舒张功能的影响。Western blotting观察磷酸化eNOS水平及Nrf2表达。结果:桂皮醛孵育显著减少改善衰老大鼠颈动脉内膜和衰老HUVEC的ROS水平,促进HUVEC中eNOS的磷酸化,增加NO水平,改善乙酰胆碱诱导的血管内皮依赖性舒张功能,对硝酸甘油诱导的非内皮依赖性舒张功能无显著的影响;Nrf2的抑制剂鸦胆子苦醇可显著阻断桂皮醛的作用。结论:桂皮醛通过Nrf2通路减少衰老相关的ROS生成,增加NO水平从而改善衰老大鼠血管内皮依赖性舒张功能。     

人脐带动脉与脐带静脉血管内皮细胞在体外相同培养环境下的定性研究

血管内皮细胞受损与许多心脑血管疾病的发生和发展有关,已成为当今生命科学、药学领域的重要的研究对象。体外分离获得血管内皮细胞以及鉴定对于研究血管功能和为心脑血管疾病建立细胞模型极为重要。该研究通过了一种简便快捷、分离纯度高的人脐带动脉内皮细胞(human umbilical cord artery endothelial cells,HUAEC)和脐带静脉内皮细胞(human umbilical cord vein endothelial cells,HUVEC)的分离及培养体系,同时从形态特征、增殖活力、成管能力、表面抗原和特异基因的表达等方面检测了二者在体外相同培养环境下的动态变化与差异。该研究发现,HUAEC和HUVEC在体外连续传代培养过程中形态特征、成管能力、表面抗原(CD144、CD31、CD309、CD133、CD34)这几个方面作为内个皮细胞的基本特性没有明显差异,尽管HUAEC相对于HUVEC增殖活力更高。对于新鲜分离的HUAEC和HUVEC,二者特异性基因表达水平具有显著差异(HUAEC高表达EFNB2、DLL4、NRP1、CXCR4;HUVEC高表达EPHB4、COUP-TF II),然而随着培养时间的延长(传代次数的增加),HUAEC丧失其特异性基因(P6)的表达之后,HUVEC仍保持其特异性基因的高表达。因此HUVEC特异性表达基因EPHB4、COUP-TF II可以作为区分体外培养的人脐带动脉或者脐带静脉来源的内皮细胞的可靠鉴定标志基因。     

低氧对血管内皮细胞肾上腺髓质素基因转录的影响

为研究低氧条件对肾上腺髓质素(adrenomedullin,ADM)基因表达的影响,本文观察了低氧引起血管内皮细胞ADM基因转录的变化,并探讨ADM在缺血缺氧性疾病机制中的作用.1 材料和方法(1)HUVEC细胞的培养人脐静脉内皮细胞株HUVEC(美国芝加哥大学Pritzker医学院Gewertz BL教授惠赠),用含20%小牛血清的M199培养基置于37℃,5%CO2培养箱内贴壁生长,待长满培养瓶培养面后用PBS冲洗2次,加入0.25%胰蛋白酶消化、传代.     

Stimulation of adhesion molecule expression in human endothelial cells (HUVEC) by adrenomedullin and corticotrophin

Adrenomedullin (AM) and corticotrophin (ACTH) are both vasoactive peptides produced by a variety of cell types, including endothelial cells. Although AM and ACTH are considered to be important in the control of blood pressure and the response to stress, respectively, their role in inflammation and the immune response has not been clarified. This study shows, with the use of a cell-based ELISA, that AM and ACTH induce cell surface expression of the adhesion molecules E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVEC). Furthermore, this effect appears to be mediated in part via elevation of cAMP, given that both peptides elevate cAMP, the cell-permeable cAMP analog dibutyryl cAMP is able to mimic induction of all three cell adhesion molecules and the effect of AM and ACTH is inhibited by the adenylyl cyclase inhibitor SQ-22536. These findings demonstrate a role for AM and ACTH in the regulation of the immune and inflammatory response. E-selectin; intercellular adhesion molecule-1; vascular cell adhesion molecule-1; adrenomedullin; adrenocorticotropic hormone; human umbilical vein endothelial cells     

Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro.

CD36 is an 88-kDa glycoprotein that has been identified on platelets, monocytes, and some endothelial cells. Experimental evidence suggests that CD36 mediates the binding of Plasmodium falciparum-infected RBC to a variety of cells, and therefore may play a role in the vascular complications associated with malaria. Additionally, CD36 may also bind the extracellular matrix proteins thrombospondin and collagen. Human umbilical vein endothelial cells have been used in in vitro models examining the binding of P. falciparum RBC to endothelial cells, but they do not consistently express cell surface CD36. Inasmuch as human dermal microvascular endothelial cells (HDMEC) differ in a variety of ways from large vessel endothelial cells, we have examined HDMEC for cell surface expression of CD36 in vivo and in vitro. Direct immunofluorescence of skin showed bright staining of HDMEC with antibody recognizing CD36 and flow cytometric analysis of cultured HDMEC revealed cell surface expression. In contrast, large vessel endothelial cells were not stained with antibody recognizing CD36 in vivo and cultured cells derived from umbilical vein failed to express cell surface CD36 in vitro. Western immunoblots of lysates of HDMEC but not human umbilical vein endothelial cells demonstrated an 88-kDa protein that comigrated with CD36 from platelets. Functional studies demonstrated that adherence of PRBC to HDMEC was inhibited up to 66% by mAb recognizing CD36. Furthermore, the expression of CD36 on HDMEC was increased in a dose- and time-dependent manner by IFN-gamma, and was decreased by protein kinase C agonists. These data demonstrate that HDMEC express functionally active CD36 and this expression can be positively and negatively regulated by soluble factors. This study demonstrates that HDMEC are useful in the study of CD36-mediated binding of PRBC to endothelial cells in vitro and provides further evidence of distinct phenotypic differences between HDMEC and large vessel endothelial cells.     

HUVEC take up opsonized zymosan particles and secrete cytokines IL-6 and IL-8 in vitro

Uptake of zymosan A particles by human umbilical vein endothelial cells (HUVEC) and its effect on cellular cytokine and oxygen radical production was examined. HUVEC took up more serum-opsonized than -unopsonized zymosan as demonstrated by flow cytometry with fluorescence-labeled particles. The former uptake was inhibited in the presence of anti-C3c antibodies and thus complement-mediated. It probably occurred via CR1 (CD35), although participation of other receptors cannot be ruled out. Scanning electron microscopy indicated that HUVEC with fully internalized zymosan particles were damaged. Prolonged incubation of both serum-opsonized and -unopsonized zymosan particles with HUVEC induced increased secretion of the proinflammatory cytokines IL-6 and IL-8 to the cell culture supernatants, but had no effect on production of oxygen radicals. The results confirm previous reports that EC can internalize yeast and other pathogens and points to complement as a mechanism of uptake, but illustrates that the cells may be damaged in the process. Moreover, EC may participate in the anti-infection defense effort by secreting proinflammatory and chemotactic cytokines in response to the contact with pathogens.     

Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages

Here, we studied the underlying mechanism of aldosterone (Aldo)-induced vascular endothelial cell damages by focusing on ceramide. We confirmed that Aldo (at nmol/L) inhibited human umbilical vein endothelial cells (HUVEC) survival, and induced considerable cell apoptosis. We propose that ceramide (mainly C18) production might be responsible for Aldo-mediated damages in HUVECs. Sphingosine-1-phosphate (S1P), an anti-ceramide lipid, attenuated Aldo-induced ceramide production and following HUVEC damages. On the other hand, the glucosylceramide synthase (GCS) inhibitor PDMP or the ceramide (C6) potentiated Aldo-induced HUVEC apoptosis. Eplerenone, a mineralocorticoid receptor (MR) antagonist, almost completely blocked Aldo-induced C18 ceramide production and HUVEC damages. Molecularly, ceramide synthase 1 (CerS-1) is required for C18 ceramide production by Aldo. Knockdown of CerS-1 by targeted-shRNA inhibited Aldo-induced C18 ceramide production, and protected HUVECs from Aldo. Reversely, CerS-1 overexpression facilitated Aldo-induced C18 ceramide production, and potentiated HUVEC damages. Together, these results suggest that C18 ceramide production mediates Aldo-mediated HUVEC damages. MR and CerS-1 could be the two signaling molecule regulating C18 ceramide production by Aldo.     

溶血卵磷脂及其受体GPR4对HUVEC的增殖及caspase3前体表达的影响

目的：研究溶血卵磷脂（lysophosphatidylcholine,LPC）及其受体GPR4（G protein-coupled receptor 4,GPR4）相互作用后对人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVEC）增殖的影响及其凋亡的诱导。方法：采用脂质体2000将装有GPR4受体基因的pEFneo真核表达载体转染HUVEC,并通过G418（800pg／ml）筛选获得GPR4基因稳定表达细胞株;RT—PCR法检测转染前后GPR4受体的mRNA水平表达情况;MTT法检测细胞的生长活力;AO染色法观察LPC对HUVEC造成损伤后形态的改变;Western blot法检测LPC与其受体相互作用后对caspase-3前体表达的影响。结果：成功获得了GPR4基因稳定表达细胞株;随着LPC浓度的增加（0．5,1,2,4,8pmoH）,抑制率分别增加了3．1％,4．4％,9．3％,17．0％,25．7％;但caspase-3前体的表达却随着浓度的增加先增加后减少。结论：LPC及其受体GPR4相互作用后可抑制HUVEC的增殖,并对HUVEC造成损伤进而诱导凋亡．使caslaase-3前体的表达先增加后减少。