过表达Syndecan-4促进人脐静脉内皮细胞增殖

目的 内皮细胞增殖在血管新生中起着非常重要的作用.本研究拟观察过表达Syndecan-4对人脐静脉内皮细胞的促增殖作用并对其相关机制进行探讨.方法 分离和培养人脐静脉内皮细胞,分别将过表达Syndecan-4的腺病毒、空病毒转染至人脐静脉内皮细胞,用免疫染色法观察人脐静脉内皮细胞的Syndecan-4的表达水平和自聚现象,用Western blotting方法检测人脐静脉内皮细胞内Akt、内皮细胞氮氧化物合酶蛋白磷酸化的水平;用磷酸化组蛋白3、5-溴脱氧尿嘧啶核苷、5-乙炔基-2′脱氧尿嘧啶核苷法观察人脐静脉内皮细胞的增殖情况.结果 与对照组相比,转染Syndecan-4组的人脐静脉内皮细胞增殖明显活跃,Syndecan-4表达明显增加,且存在自聚现象;Western blotting检测发现,Akt、内皮细胞氮氧化物合酶磷酸化水平较对照组明显升高.结论 过表达人脐静脉内皮细胞Syndecan-4可诱导Syndecan-4自聚,并可能通过活化下游Akt、内皮细胞氮氧化物合酶,促进人脐静脉内皮细胞的增殖.     

糖基化终产物对人脐静脉内皮细胞一氧化氮合酶活性和表达的影响及二甲双胍的保护作用

目的 探讨糖基化终产物及二甲双胍对人脐静脉内皮细胞一氧化氮合酶活性和表达的影响.方法 用胶原酶法分离人脐静脉内皮细胞并加以培养.将内皮细胞与不同浓度的糖基化终产物和二甲双胍分别孵育3、6、12、24 h,CCK-8法测定人脐静脉内皮细胞增殖活性.硝酸还原酶法测定一氧化氮含量,分光光度法测定一氧化氮合酶活性,蛋白免疫印迹法检测内皮型一氧化氮合酶蛋白表达水平.结果 糖基化终产物抑制人脐静脉内皮细胞增殖,二甲双胍促进人脐静脉内皮细胞增殖.糖基化终产物抑制人脐静脉内皮细胞的一氧化氮生成和一氧化氮合酶活性(P<0.01),呈剂量、时间依赖关系.二甲双胍(与对照组相比)或与糖基化终产物共同干预(与糖基化终产物组相比)均增加人脐静脉内皮细胞一氧化氮生成和一氧化氮合酶活性(P<0.01).糖基化终产物与人脐静脉内皮细胞共同孵育24 h后,内皮型一氧化氮合酶表达水平明显下降;二甲双胍上调内皮型一氧化氮合酶的表达;与糖基化终产物组相比,糖基化终产物与二甲双胍共同干预组内皮型一氧化氮合酶表达上调(P<0.01).结论 二甲双胍能够改善糖基化终产物导致的人脐静脉内皮细胞损伤.     

醛固酮对人脐静脉内皮细胞血管内皮生长因子表达的影响

目的醛固酮对人脐静脉内皮细胞增殖的影响及其机制。方法应用MTT法测试细胞的生长曲线,检测不同浓度的醛固酮对人脐静脉内皮细胞的增殖,流式细胞仪检测醛固酮对人脐静脉内皮细胞的凋亡,半定量RT-PCR及Western印迹分别检测人脐静脉内皮细胞的血管内皮生长因子mRNA水平及蛋白水平的表达。结果①MTT比色法显示,醛固酮对人脐静脉内皮细胞增殖具有抑制作用且呈浓度依赖性;②当醛固酮浓度增加到800μg/L后,其增殖抑制率不再升高,而此浓度时人脐静脉内皮细胞的凋亡率达到峰值的26.5%±3.3%;③经过醛固酮处理,人脐静脉内皮细胞的血管内皮生长因子mRNA及蛋白水平的表达量均显著下降。结论醛固酮通过降低人脐静脉内皮细胞的血管内皮生长因子表达抑制人脐静脉内皮细胞增殖并促进人脐静脉内皮细胞凋亡。     

内皮型一氧化氮合酶基因启动子区DNA甲基化

目的 探讨内皮型一氧化氮合酶基因启动子区DNA甲基化在同型半胱氨酸致内皮细胞损伤中的作用机制.方法 原代培养人脐静脉内皮细胞,加入0、50、100、200、500 μmol/L同型半胱氨酸和100 μmol/L同型半胱氨酸+维生素B12+叶酸的培养液中孵育72 h.MTT法检测人脐静脉内皮细胞的增殖活性;实时定量PCR测定内皮型一氧化氮合酶mRNA表达;化学比色法测定内皮型一氧化氮合酶活性;硝酸还原酶法检测一氧化氮生成量.巢式降落式甲基化特异性PCR法检测内皮型一氧化氮合酶基因启动子区DNA甲基化改变;同位素法检测DNA甲基化转移酶的活性.结果 人脐静脉内皮细胞与不同浓度同型半胱氨酸孵育72 h后,人脐静脉内皮细胞的增殖活性降低,内皮型一氧化氮合酶mRNA表达、内皮型一氧化氮合酶活性和一氧化氮的含量明显下降.内皮型一氧化氮合酶基因启动子序列DNA甲基化程度随同型半胱氨酸浓度的升高而增加,且DNA甲基化转移酶活性升高,而叶酸和维生素B12有一定拮抗作用,与对照组比较差异有显著性(P＜0.05和P＜0.01).结论 同型半胱氨酸可导致内皮型一氧化氮合酶基因启动子区序列高甲基化修饰,并出现相应的内皮型一氧化氮合酶基因表达下调,这可能是同型半胱氨酸致内皮细胞损伤的重要机制之一.     

MicroRNA-99a促进新生小鼠心肌细胞增殖

目的有效地促进心肌细胞增殖为亟待发展的治疗策略。本研究拟观察高表达microRNA-99a（miR-99a）对体外培养心肌细胞增殖的影响并对其相关机制进行探讨。方法利用高表达miR-99a的慢病毒载体转染体外培养的乳鼠心肌细胞，诱导其在乳鼠心肌细胞中高表达。采用噻唑蓝法、EDU法检测乳鼠心肌细胞增殖能力Westernblot检测乳鼠心肌细胞ERK1/2蛋白表达及其蛋白磷酸化水平。结果miR-99a在乳鼠心肌细胞高表达后，噻唑蓝法、EDU法检测结果均表明:miR-99a转染组细胞增殖能力明显高于空病毒转染对照组（P<0.05）。Westernblot检测发现miR-99a病毒转染组ERK1/2蛋白磷酸化水平较对照组明显升高（P<0.05）。结论miR-99a高表达能促进乳鼠心肌细胞增殖，其机制可能部分通过激活Erk1/2通路。     

内含子源性miRNA通过转录因子AP1调节内皮型一氧化氮合酶表达及血管内皮细胞增殖作用

目的　前期工作发现,内皮型一氧化氮合酶（eNOS）第4内含子的27碱基重复子产生相应27-nt　miRNA,并对eNOS的转录和表达有负反馈调节作用。进一步探讨该内含子源性27-nt　miRNA对内皮型一氧化氮合酶基因调节的分子机制及血管内皮细胞增殖相关转录因子AP1的作用机制。方法　应用MTT法检测人脐静脉内皮细胞（HUVEC）的增殖抑制率;细胞划痕实验检测HUVEC迁移能力;免疫组织化学方法检测内皮细胞中eNOS和AP1蛋白表达水平;进一步用Western　Blot检测27-nt　miRNA表达相关情况及细胞核转录因子eNOS表达情况。结果　27-nt　miRNA对HUVEC增殖具有强烈抑制作用　（P<0.05）;27-nt　miRNA对HUVEC迁移具有显著抑制作用（P<0.05）;27-nt　miRNA显著降低eNOS和AP1蛋白表达（P<0.05）;27-nt　miRNA通过负调控AP1降低eNOS蛋白表达（P<0.05）。结论　27-nt　miRNA显著抑制eNOS和AP1蛋白表达,AP1在27-nt　miRNA对eNOS表达调节中起重要作用。     

内皮型一氧化氮合酶与心肌缺血再灌注损伤的关系研究进展

内皮型一氧化氮合酶（eNOS）参与心肌缺血再灌注损伤（MIRI）调控机制和信号通路，其不仅在内皮细胞中表达，也在心肌细胞、肥大细胞、肾上皮细胞、红细胞、白细胞和血小板中表达；eNOS既可以合成NO，扩张血管，也可以合成超氧化物，加剧氧化应激。在MIRI中抑制eNOS解偶联和增加其有益磷酸化，可保护血管内皮功能、抑制氧化应激、逆转凋亡和减轻炎症反应等；eNOS可通过磷脂酰肌醇3激酶/丝氨酸/苏氨酸蛋白激酶/eNOS、沉默信息调节剂1/eNOS、腺苷单磷酸活化蛋白激酶/eNOS/一氧化氮等信号通路在MIRI中发挥重要作用。     

高糖影响内皮细胞功能不良机制的初步探究

目的探讨高糖对人脐静脉内皮细胞(HUVEC)功能不良的机制。方法分离并培养HUVEC,取第3代细胞分为正常对照组、甘露醇对照组(33mmol/L)和高糖对照组(33mmol/L),处理36h后,用RT-PCR和Western blot测哺乳动物雷帕霉素靶蛋白雷帕霉素不敏感配体(RICTOR)表达情况。转染RICTOR过表达腺病毒为高糖腺病毒组,转染空白病毒AD-GFP作为高糖空病毒组,检测蛋白激酶B(Akt)和内皮型一氧化氮合酶(eNOS)的磷酸化水平,用硝酸还原酶法测定NO释放量。结果高糖对照组RICTOR蛋白表达量较正常对照组显著降低(1.00±0.16 vs 2.69±0.07,P<0.01)。与高糖空病毒组比较,高糖腺病毒组RICTOR表达增加(0.57±0.03 vs0.29±0.02,P<0.01),磷酸化Akt-Ser473(0.95±0.05 vs 0.56±0.04,P<0.01)及磷酸化eNOS-Ser1177增加(0.97±0.05 vs 0.55±0.07,P<0.01),内皮细胞NO释放增多(0.85±0.06 vs 0.56±0.04,P<0.05)。结论纠正RICTOR表达能改善高糖诱发的血管内皮细胞功能不良。     

罗格列酮对高胰岛素培养的内皮细胞一氧化氮的影响及其机制研究

目的观察罗格列酮（RGZ）对高胰岛素培养的人脐静脉内皮细胞（HUVEC）NO浓度和内皮型一氧化氮合酶（eNOS）、磷酯酰肌醇3激酶（P13K）和蛋白激酶B（PKB）表达的影响,探讨RGZ改善高胰岛素状态下内皮功能障碍的信号转导机制。方法高浓度胰岛素培养HUVEC72h,并用不同浓度的RGZ进行干预。检测NO浓度,PI3K mRNA的表达,PKB、eNOS总蛋白和PKB丝氨酸473（PKB-Ser473）、eNOS丝氨酸1177（eNOS-Ser1177）的磷酸化表达。结果高浓度胰岛素培养HUVEC能呈剂帚和时间依赖性地降低N0的浓度,抑制内皮细胞P13KmRNA表达和PKB-Ser473、eNOS-Ser1177的磷酸化。用RGZ干预能硅著升高高胰岛素培养的内皮细胞NO的浓度和PKB、eNOS的磷酸化,增强PI3KmRNA表达;eNOS和P13K阻断剂均能阻断RGZ对高胰岛素培养的内皮细胞中NO浓度的升高,PI3K阻断剂还能阻断RGZ对高胰岛素培养内皮细胞PKB、eNOS的磷酸化。结论高胰岛素能下调P13K／PKB／eNOs信号通路而抑制内皮细胞NO的产生,RGZ能通过上调PI3K／PKB通路而增强高胰岛素培养的内皮细胞eNOS的活性和NO的产生。     

不同剂量甲醛对人脐静脉内皮细胞一氧化氮含量的影响

目的观察不同剂量甲醛对人脐静脉内皮细胞一氧化氮含量的影响,分析甲醛诱导内皮细胞损伤在心血管疾病发病中的意义。方法将人脐静脉内皮细胞株分别与不同浓度甲醛(分别为0、5、10、20、40和80μmol/L)的DMEM培养基孵育,同时以过氧化氢(100μmol/L)为阳性对照组,抗氧化剂维生素C(100mg/L)为保护组。共培养24h后,分光光度法检测乳酸脱氢酶漏出量;硫代巴比妥法检测细胞上清液中丙二醛浓度;硝酸还原酶法测定培养液中一氧化氮浓度;生化分析法测定内皮细胞一氧化氮合酶活性;免疫细胞化学法测定内皮型一氧化氮合酶和诱导型一氧化氮合酶的蛋白表达;RT-PCR法测定内皮型一氧化氮合酶和诱导型一氧化氮合酶的mRNA表达。结果不同浓度甲醛呈剂量依赖性地促进细胞乳酸脱氢酶漏出,促进内皮细胞上清液丙二醛和一氧化氮的产生;降低内皮型一氧化氮合酶的活性,抑制内皮型一氧化氮合酶在蛋白和基因水平的表达;增加诱导型一氧化氮合酶活性,促进诱导型一氧化氮合酶在蛋白和基因水平的表达。结论甲醛诱导人脐静脉内皮细胞损伤,机制可能与其抑制内皮型一氧化氮合酶的活性及表达,诱导诱导型一氧化氮合酶的活性及表达,从而诱导内皮细胞产生过多的一氧化氮有关;维生素C通过降低一氧化氮产生,减轻甲醛诱导的内皮细胞损伤。     

Integrin-linked kinase overexpression promotes epithelial-mesenchymal transition via nuclear factor-κB signaling in colorectal cancer cells

AIM: To investigate the effect of integrin-linked kinase(ILK) on proliferation, metastasis, and invasion of the colorectal cancer cell line SW480. METHODS: In this study, the colorectal cancer cell line SW480 was stably transfected with ILK plasmids, and small interfering RNA(si RNA) was used to knockdown expression of nuclear factor(NF)-κB/p65. Methylthiazole tetrazolium(MTT) assay was performed to measure proliferation, and the wound healing migration assay and matrigel invasion assay were used to test the metastasis and invasion ability of SW480 cells. To explore the epithelial-mesenchymal transition(EMT) process, embryonic development, and the invasion and metastasis of tumors, the protein level of E-cadherin, vimentin, snail, and slug was detected by western blot. Immunofluorescence was also used to detect E-cadherin expression. Western blot was used to determine the level of phosphorylated-inhibitor of kappa B(IκB)a, inhibitor of gamma B(IγB)a, and nuclear factor kappa B(NF-κB) expressions and toexplore the ILK signaling pathway. RESULTS: Western blot results revealed that ILK expression significantly increased when ILK was overexpressed in SW480 cells(P 0.05). Proliferation, metastasis, and invasion ability were improved in the vector-ILK group compared to the vector group(P 0.05). Immunofluorescence results revealed that E-cadherin fluorescence intensity decreased after ILK was overexpressed(P 0.05). Western blot results revealed that the protein expression of E-cadherin was reduced, while vimentin, snail, and slug were upregulated when ILK was overexpressed in SW480 cells(P 0.05). In order to determine the role of the NF-κB signaling pathway in ILK overexpression promoted EMT occurrence, we overexpressed ILK in SW480 cells and found that levels of NF-κB/p65 and cytoplasmic phosphorylated-IκBa were increased and that cytoplasmic IкBa levels were decreased compared to the control group(P 0.05). Furthermore, NF-κB/p65 knockout revealed that E-cadherin was increased in the overexpressed ILK group. CONCLUSION: ILK overexpression improved the proliferation, metastasis, and invasion ability of SW480 cells, and this effect may be mediated by the NF-κB signaling pathway.     

Pharmacological implications from the adhesion-induced signaling profiles in cultured human retinal pigment epithelial cells

Extracellular matrix (ECM) plays an active and complex role in regulating cellular behaviors, including proliferation and adhesion. This study aimed at delineating the adhesion-induced signaling profiles in cultured human retinal pigment epithelium (RPE) cells and investigating the antiadhesion effect of antiproliferative drugs in this context. RPE R-50 cells grown on various ECM molecules, such as type I and IV collagens, fibronectin, and laminin, were used for adhesion assay and for examining the phosphorylation profiles of signaling mediators including Akt, extracellular signal-regulated kinase (ERK) 1/2, and integrin-linked kinase (ILK) using Western blotting. The cells receiving antiproliferative drug treatment at subtoxic doses were used to evaluate their antiadhesive and suppressive effects on kinase activities. ECM coating enhanced adhesion and spreading of RPE cells significantly. The cellular attachment onto ECM-coated surfaces differentially induced Akt, ERK1/2, and ILK phosphorylation, and concomitantly increased p53 phosphorylation and cyclin D1 expression, but decreased Bcl-2/Bax ratios. Treatment with antiproliferative agents, including 5-fluorouracil, mitomycin C, and daunomycin, at subtoxic doses suppressed the ability of RPE cells to adhere to ECM substratum significantly. This suppression was in part mediated through reduction of integrin β1 and β3 expressions and interfering Akt-ILK signaling activity. Mechanistically, blockade of PI3K/Akt signaling resulted in the suppressed adhesion of RPE cells to ECM. These findings support the hypothesis that, in addition to their antimitogenic effect, antiproliferative agents also exhibit suppressive effect on the adhesiveness of cultured RPE cells. Moreover, inhibitors of the PI3K/Akt signaling mediator can potentially be used as therapeutic agents for proliferative vitreoretinopathy.     

Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells

PTEN is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. Somatic mutations of PTEN are found in a number of human malignancies, and loss of expression, or mutational inactivation of PTEN, leads to the constitutive activation of protein kinase B (PKB)/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We recently have demonstrated that the integrin-linked kinase (ILK) can phosphorylate PKB/Akt on Ser-473 in a phosphoinositide phospholipid-dependent manner. We now demonstrate that the activity of ILK is constitutively elevated in a serum- and anchorage-independent manner in PTEN-mutant cells, and transfection of wild-type (WT) PTEN into these cells inhibits ILK activity. Transfection of a kinase-deficient, dominant-negative form of ILK or exposure to a small molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473, but not on Thr-308, in the PTEN-mutant prostate carcinoma cell lines PC-3 and LNCaP. Transfection of dominant-negative ILK and WT PTEN into these cells also results in the inhibition of PKB/Akt kinase activity. Furthermore, dominant-negative ILK or WT PTEN induces G(1) phase cycle arrest and enhanced apoptosis. Together, these data demonstrate a critical role for ILK in PTEN-dependent cell cycle regulation and survival and indicate that inhibition of ILK may be of significant value in PTEN-mutant tumor therapy.     

人剪切修复基因着色性干皮病基因D对人脐静脉内皮细胞的促凋亡作用

目的探讨人剪切修复基因着色性干皮病基因D(XPD)对人脐静脉内皮细胞(HUVEC)的促凋亡作用。方法用脂质体转染法瞬时转染HUVEC,转染重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2,并用未转染的与重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2具有相同遗传背景和代数的HUVEC作为空白对照。实验分为3组:正常对照组、pEGFP-N2组和pEGFP-N2/XPD组。用荧光显微镜观察绿色荧光蛋白报告基因表达情况,用流式细胞仪检测细胞凋亡情况,用RT-PCR和Western Blot检测XPD、Bcl-2、Bax和wt-p53表达量的变化,用MTT法观察细胞增殖活力。结果在荧光显微镜下,可在转染了重组质粒pEGFP-N2/XPD或空载质粒pEGFP-N2的细胞中观察到绿色荧光,即转染成功;流式细胞仪结果显示,重组质粒pEGFP-N2/XPD的转染引起细胞凋亡增加(P<0.05或P<0.01);RT-PCR和Western Blot检测发现,重组质粒pEGFP-N2/XPD的转染使得XPD表达增高(P<0.05),同时使得Bcl-2表达降低,Bax和wt-p53表达增高(P<0.05或P<0.01);MTT结果显示,重组质粒pEGFP-N2/XPD的转染抑制细胞增殖活力(P<0.05)。结论 XPD能促进HUVEC凋亡,下调XPD的表达,有望成为治疗动脉粥样硬化的一个新靶点。     

CREG regulates vascular endothelial cell migration mediated by ILK-β-parvin signal pathway

Background To investigate the effects and mechanisms of cellular repressor of ElA stimulated genes (CREG) on endothelial cell(EC) migration.Methods vascular endothelial cells(VE),CREG overexpression VEs, CREG suppression VEs and VEs transfected with CREG gene modified adenovirus(Ad-CREG) were cultured with dulbecco’s modified eagle’s medium contained 10%fetal calf serum. Western blot was used to detect the protein level of CREG and integrin-linked kinase(ILK) in the four kind ECs.Tran-swell migration model was applied to compare the migration cell number of the four kind ECs.Two kinds of ILK mutant plasmids;PCXN2-flag-ILK wt-IRES-GFP(wild-type ILK)and PCXN2-flag-ILK p-parvin-IRES-GFP(P-parvin-binding mutant) were used to transfect VS and VE respectively,then the two kind transfection ECs were named as VS-wtILK and VE-P -parvin which were selected by G418(600ng/ml)for 2 weeks;Transwell migration model was applied to compare migration capability before and after ILK plasmids transfecting VE and VS.Results Western blot analysis showed that CREG overexpression promoted ILK expression in ECs,on the contrary,ILK expression was down-regulated in CREG silent ECs(P<0.05).Further more,ILK expression was up-regulated obviously in VE transfected with Ad-CREG(P< 0.05);Transwell migration model showed that EC’s migration capability was positively correlated with the expression level of CREG in EC,that is,CREG overexpression induced VE migration and CREG silent suppressed VE migration, moreover,Ad-CREG transfecting VE showed better migration capability accompanied with CREG expression increase by transwell migration model(P<0.05).In order to know the relationship between ILK expression and cell migration,we obtained stable transfection cell strains of VS-wtILK and VE-Pparvin, transwell migration model demonstrated that VS-wtILK remarkably corrected the poor migration capability of VS(P< 0.01),butβ-parvin combining site mutation in ILK genes inhibited VE migration markedly(P<0.01).Conclusions ILKp -parvin     

RGD peptides confer survival to hepatocytes via the beta1-integrin-ILK-pAkt pathway

BACKGROUND/AIMS: Allogeneic cell transplantation is characterized by a lack of sustained survival of the transplanted cells in the recipient. Activation of the appropriate integrin-linked signaling pathways in cells can promote cell survival. The purpose of this study was to determine how presence or absence of anti-beta1 integrin chain antibodies or RGD peptides affects the survival of hepatocytes. METHODS: Hepatocytes of BN rats were isolated. Hepatocyte survival was tested after the hepatocytes had been cultured in the presence of anti-beta1 integrin antibodies or RGD peptides. Hepatocytes that had been given a different treatment were stained for caspase 3 (apoptosis marker) and phospho-Akt Ser 473 (survival marker) and were measured for their integrin-linked kinase (ILK) activity. RESULTS: Ligation of integrins using antibodies against the beta1 integrin chain or RGD peptides protected isolated hepatocytes from apoptosis and resulted in an increased ILK activity and persistent phosphorylation of protein kinase B/Akt at serine 473. CONCLUSIONS: Integrin activation in isolated hepatocytes contributes to the activation of ILK, phosphorylation of Akt on serine residue 473, and inhibition of apoptosis. Integrin signaling through the ILK-phospho Akt pathway protects isolated hepatocytes from apoptosis. This notion may potentially be applied to render the transplantation of hepatocytes more effective.     

脂联素对内皮祖细胞增殖和迁移能力的影响

目的研究脂联素通过磷酸肌醇3激酶/蛋白激酶B(Akt)通路改善内皮祖细胞(EPC)增殖、迁移能力的影响并探讨其可能机制。方法利用密度梯度离心法分离、培养人外周血单个核细胞,将EPC分为对照组、脂联素组(10μg/ml)、磷酸肌醇3激酶抑制剂干预组(干预1组)和细胞外信号调节蛋白激酶(ERK)抑制剂干预组(干预2组)。采用四唑盐比色法、细胞集落形成单位计数等方法观察各组EPC增殖能力的变化情况,应用transwell小室法分析EPC迁移能力,采用免疫蛋白印迹法观察脂联素处理EPC后,Akt、磷酸化Akt、ERK、磷酸化ERK的表达情况。结果脂联素组EPC增殖能力较对照组明显提高(P<0.05),干预1组EPC增殖、迁移效应较脂联素组明显抑制(P<0.05),而干预2组对EPC增殖、迁移改善效应无明显影响。与对照组比较,脂联素组磷酸化Akt表达明显增加,干预1组磷酸化Akt表达及脂联素组磷酸化ERK表达无明显增加。结论脂联素具有促进EPC增殖、迁移等功能活性的作用,其主要机制可能与磷酸肌醇3激酶/Akt信号通路激活有关。     

基于自噬与凋亡平衡探讨补肾活血汤对衰老耳蜗毛细胞株HEI-OC1的保护作用

目的 探讨补肾活血汤对衰老耳蜗毛细胞株HEI-OC1自噬和凋亡的影响.方法 应用D-半乳糖构建衰老HEI-OC1细胞的体外模型后,将细胞分为对照组、衰老组、含药血清低、中及高剂量组(5％、10％、20％),采用噻唑蓝(MTT)比色法检测耳蜗HEI-OC1细胞的增殖能力;β-半乳糖苷酶染色检测细胞衰老状态;膜联蛋白V/碘...     

miR-20a-5p通过靶向MRTFA缓解ox-LDL诱导的内皮细胞损伤

目的探讨miR-20a-5p是否可通过靶向心肌素相关转录因子A(MRTFA)缓解氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)损伤。方法 RT-qPCR检测miR-20a-5p在不同剂量不同时间oxLDL诱导的HUVEC中的表达; MTT法、流式细胞术和Western blot检测过表达miR-20a-5p和MRTFA对ox-LDL诱导HUVEC增殖和凋亡的影响;乳酸脱氢酶(LDH)测试盒测定过表达miR-20a-5p对ox-LDL诱导HUVEC中LDH释放的影响; ELISA法检测过表达miR-20a-5p和MRTFA对ox-LDL处理的HUVEC中氧化应激指标超氧化物歧化酶(SOD)、一氧化氮(NO)、内皮型一氧化氮合酶(e NOS)和丙二醛(MDA)的影响;荧光素酶报告和Western blot实验验证miR-20a-5p与MRTFA的靶向关系。结果 miR-20a-5p的表达随着ox-LDL诱导时间和剂量的增加而逐渐下降;过表达miR-20a-5p可部分逆转ox-LDL诱导对HUVEC增殖和凋亡的影响;上调miR-20a-5p可部分修复ox-LDL诱导对HUVEC氧化应激损伤; MRTFA是miR-20a-5p的靶基因;在ox-LDL诱导HUVEC中,MRTFA过表达可逆转miR-20a-5p对ox-LDL诱导HUVEC细胞增殖和凋亡、氧化应激损伤指标的影响。结论 miR-20a-5p靶向MRTFA修复ox-LDL诱导的HUVEC损伤。     

别嘌呤醇对同型半胱氨酸和低密度脂蛋白协同损伤血管内皮细胞的保护作用

目的:探讨别嘌呤醇(Allo)对同型半胱氨酸(Hcy)和低密度脂蛋白(LDL)协同损伤人脐静脉血管内皮细胞的保护作用。方法:①将人脐静脉血管内皮细胞与一定浓度的Hcy、LDL及Allo共同培养24小时,分为Hcy(H)组、Hcy+Allo(H+A)组、LDL(L)组、Allo(A)组、LDL+Allo(L+A)组、Hcy+LDL(H+L)组、Hcy+LDL+Allo(H+L+A组,其中Allo在MTT试验中的浓度分别为0.1、0.2、0.3mmol/l)及正常对照组(各n=6孔),应用MTT试验观察Allo是否对其有保护作用。②测定细胞培养上清液乳酸脱氢酶、丙二醛和一氧化氮含量,分别观察内皮细胞损伤、脂质过氧化程度及血管舒张因子产生情况(H+L+A组中Allo为0.2mmol/L)。③同时,利用半定量逆转录聚合酶链式反应方法,检测内皮型一氧化氮合酶转录水平,以观察Allo是否影响其的转录。结果:H+L组、H+A组、H组、H+L+A组(Allo0.1～0.3mmol/L)细胞与正常对照组相比,吸光度减小,分泌一氧化氮减少,乳酸脱氢酶和丙二醛增加,有显著性差异(P<0.05)。除H+L+A组(Allo0.1mmol/L)外,其余各组细胞吸光度较H+L组均明显升高,有显著性差异(P<0.05)。H+L+A组(Allo0.1～0.3mmol/L)细胞吸光度随着Allo浓度增加而增加。除H组外,余各组与H+L组比较一氧化氮生成量均增加,有显著性差异(P<0.05)。与H+L组比较,其余各组细胞损伤、脂质过氧化程度均减轻,有显著性差异(P<0.05)。内皮型一氧化氮合酶的半定量逆转录聚合酶链式反应结果显示各组间的产物电泳带无显著差异。结论:Allo对Hcy和LDL协同损伤人脐静脉血管内皮细胞有保护作用,提示其抗动脉粥样硬化的作用。