PI16重组腺病毒表达载体的构建及其在人主动脉瓣间质细胞中的表达

目的构建携带人肽酶抑制蛋白16(PI16)基因的重组腺病毒表达载体,并研究其在人主动脉瓣间质细胞(hAVICs)中的表达。方法合成带有EcoRⅠ酶切位点的PI16引物,PCR扩增PI16基因,将目的基因克隆到穿梭载体pAV-MCMV-GFP-3FLAG中,PCR及测序鉴定穿梭质粒pAV-MCMV-PI16-GFP-3FLAG。将穿梭质粒与辅助质粒pBHGlox(delta)E1,3Cre共转染HEK 293细胞,获取重组腺病毒Ad-MCMV-PI16-GFP-3FLAG。转染HEK 293细胞大量扩增后,采用CsCl梯度离心法纯化病毒,半数组织培养感染剂量(TCID50)法测定病毒滴度。用Ad-MCMV-PI16-GFP-3FLAG感染hAVICs,Western blot检测PI16在hAVICs中的表达。结果成功构建了表达PI16蛋白的重组腺病毒,滴度达到5.01×1010 TCID50/ml,转染hAVICs后能高效地表达PI16蛋白。结论成功构建携带人PI16基因的重组腺病毒,为进一步研究PI16基因的作用提供了基础。     

主动脉瓣膜间质细胞分化机制研究进展

瓣膜纤维化或钙化是瓣膜性心脏病(VHD)的两大重要病理表现,其中瓣膜间质细胞(VICs)由成纤维细胞样表型分化为肌成纤维细胞表型或成骨细胞表型,是瓣膜发生早期病理改变的关键环节。VHD多累及主动脉瓣,近年来有关VICs分化的研究多聚焦于主动脉瓣来源细胞。本文就主动脉瓣VICs分化的最新研究进展予以综述,旨在为VHD的防治研究提供更为全面的参考。     

经导管主动脉瓣置换术的研究进展

主动脉瓣狭窄(AS)是老年人最常见的瓣膜退行性改变,发病率正逐年上升.经导管主动脉瓣置换术(TAVR)的临床应用为AS患者提供了一种全新的治疗方法 ,目前TAVR已经成为不能进行外科手术或具有高外科手术风险的重度AS患者的有效替代治疗方案.欧美国家开展的TAVR注册研究进一步肯定了TAVR的效果.近期一些重要的随机对照临床试验及注册研究结果 已经报道,现将近期重要的试验结果 作一综述.     

老年钙化性主动脉瓣狭窄与外周血炎性细胞及血脂水平的关系

摘要： 目的 探讨老年 （≥65岁） 患者发生钙化性主动脉瓣狭窄与其外周血炎性细胞水平及脂质代谢异常的相关性。方法 连续纳入2015年6月—2017年6月间在我院诊断为钙化性主动脉瓣狭窄的老年患者76例作为病例组,选取78例因胸部不适住院的老年人 （除外心脏瓣膜病） 作为对照组。2组入院时均检测白细胞计数 （WBC）、 中性粒细胞比例 （N%）、 中性粒细胞计数 （N）、 淋巴细胞计数 （L）、 中性粒细胞淋巴细胞比值 （NLR）、 超敏C-反应蛋白 （hs- CRP）、 氨基末端B型利钠肽原 （NT-pro BNP）、 总胆固醇 （TC）、 三酰甘油 （TG）、 载脂蛋白α （apo-α）、 高密度脂蛋白（HDL）、 低密度脂蛋白 （LDL）、 极低密度脂蛋白 （VLDL） 等指标, 比较这些指标在2组间的差异。结果 病例组WBC、 N%、 N、 NLR、 hs-CRP、 NT-pro BNP、 VLDL均高于对照组, L、 HDL均低于对照组 （P＜0.05）; 多因素回归分析显示, 吸烟、 hs-CRP及NT-pro BNP升高是老年钙化性主动脉瓣狭窄的独立危险因素。结论 老年钙化性主动脉瓣狭窄不是一种简单的退行性病变, 其与全身炎症反应、 脂质代谢异常有关。     

主动脉瓣钙化与冠心病的关系研究

目的探讨主动脉瓣钙化与冠心病发病之间的关系。方法回顾性研究同期行冠状动脉造影检查和超声心动图检查患者653例,并对所有患者分组：正常对照组和主动脉瓣钙化组（左冠瓣钙化、右冠瓣钙化,主动脉瓣多瓣膜钙化）,对比研究主动脉瓣钙化组与正常对照组间冠心病发病率差异,同时比较单瓣主动脉瓣钙化与冠状动脉狭窄是否发生于同侧。结果主动脉瓣钙化组冠心病的检出率明显高于正常对照组,单瓣主动脉瓣钙化与同侧冠脉狭窄无明显相关性。结论主动脉瓣钙化患者有更高的冠心病发病率,主动脉瓣钙化可以作为冠心病无创评估的一个参考指标。     

Ramipril retards development of aortic valve stenosis in a rabbit model: mechanistic considerations

BACKGROUND AND PURPOSE

Aortic valve stenosis (AVS) is associated with significant cardiovascular morbidity and mortality. To date, no therapeutic modality has been shown to be effective in retarding AVS progression. We evaluated the effect of angiotensin-converting enzyme inhibition with ramipril on disease progression in a recently developed rabbit model of AVS.

EXPERIMENTAL APPROACH

The effects of 8 weeks of treatment with either vitamin D₂ at 25 000 IU for 4 days a week alone or in combination with ramipril (0.5 mg·kg⁻¹) on aortic valve structure and function were examined in New Zealand white rabbits. Echocardiographic aortic valve backscatter (AV_BS) and aortic valve : outflow tract flow velocity ratio were utilized to quantify changes in valve structure and function.

KEY RESULTS

Treatment with ramipril significantly reduced AV_BS and improved aortic valve : outflow tract flow velocity ratio. The intravalvular content of the pro-oxidant thioredoxin-interacting protein was decreased significantly with ramipril treatment. Endothelial function, as measured by asymmetric dimethylarginine concentrations and vascular responses to ACh, was improved significantly with ramipril treatment.

CONCLUSIONS AND IMPLICATIONS

Ramipril retards the development of AVS, reduces valvular thioredoxin-interacting protein accumulation and limits endothelial dysfunction in this animal model. These findings provide important insights into the mechanisms of AVS development and an impetus for future human studies of AVS retardation using an angiotensin-converting enzyme inhibitor.     

Inhibitory effect of Uncaria sinensis on human aortic smooth muscle cell migration is based on matrix metalloproteinase-9 inhibitory activity

Medicinal extracts of Cho-Deung-san and Uncaria sinensis Havil. (UR) have previously been shown to have inhibitory effects on migration of vascular smooth muscle cells (VSMC) and matrix metalloproteinase (MMP)-2/9 production, which play key roles in the development of atherosclerosis. In this study, we have more extensively investigated the inhibitory effect of UR on MMP-9 activity and TNF- induced human aortic smooth muscle cells (HASMC) migration. The result from gelatin zymography showed that UR inhibited MMP-9 activity in a dose-dependent manner (IC₅₀ = 55 μg/ml). In addition, UR strongly inhibited the migration of HASMC induced by TNF- treatment (IC₅₀ = 125 μg/ml), although it has very low cytotoxic effect on HASMC (IC₅₀ > 500 μg/ml). These results suggest that UR is a potential anti-atherosclerotic agent through inhibition of MMP-9 activity and VSMC migration.     

Vitamin D(2) supplementation induces the development of aortic stenosis in rabbits: interactions with endothelial function and thioredoxin-interacting protein

Understanding of the pathophysiology of aortic valve stenosis (AVS) and finding potentially effective treatments are impeded by the lack of suitable AVS animal models. A previous study demonstrated the development of AVS in rabbits with vitamin D(2) and cholesterol supplementation without any hemodynamic changes in the cholesterol supplemented group alone. The current study aimed to determine whether AVS develops in an animal model with vitamin D(2) supplementation alone, and to explore pathophysiological mechanisms underlying this process. The effects of 8 weeks' treatment with vitamin D(2) alone (n=8) at 25,000 IU/4 days weekly on aortic valve structure and function were examined in male New Zealand white rabbits. Echocardiographic aortic valve backscatter (AV(BS)), transvalvular velocity, and transvalvular pressure gradient were utilized to quantitate changes in valve structure and function. Valvular histology/immunochemistry and function were examined after 8 weeks. Changes in valves were compared with those in endothelial function and in valvular measurement of thioredoxin-interacting protein (TXNIP), a marker/mediator of reactive oxygen species-induced oxidative stress. Vitamin D(2) treated rabbits developed AVS with increased AV(BS) (17.6+/-1.4 dB vs 6.7+/-0.8 dB, P<0.0001), increased transvalvular velocity and transvalvular pressure gradient (both P<0.01 via 2-way ANOVA) compared to the control group. There was associated valve calcification, lipid deposition and macrophage infiltration. Endothelial function was markedly impaired, and intravalvular TXNIP concentration increased. In this model, vitamin D(2) induces the development of AVS with histological features similar to those of early AVS in humans and associated endothelial dysfunction/redox stress. AVS development may result from the loss of nitric oxide suppression of TXNIP expression.     

阿司匹林对人主动脉血管内皮细胞炎性损伤的保护作用

目的：研究阿司匹林（aspirin,Asp）对脂多糖（lipopolysaccharide,LPS）诱导人主动脉内皮细胞（human aortic endothelial cells,HAECs）损伤的保护作用,并进一步阐明其对一氧化氮合酶（NOS）及血管内皮生长因子（VEGF）及其相关受体信号的调控。方法：LPS建立HAECs损伤模型。苏木精-伊红（HE）染色观察细胞形态;MTT法、划痕实验分析HAECs损伤修复能力;ELISA测定一氧化氮（NO）含量;Western blot检测内皮型一氧化氮合酶（eNOS）、诱导型一氧化氮合酶（iNOS）、VEGF和血管内皮生长因子受体-2（VEGFR-2）蛋白表达。结果：给药12 h后Asp明显改善LPS（5 mg·L^-1）导致的细胞损伤、提高修复能力（P<0.05）,并上调NO分泌量及VEGF、VEGFR-2的蛋白表达（P<0.01）;升高eNOS蛋白的表达（P<0.01）。而给药24 h后阿司匹林显著下调LPS导致的NO分泌量及iNOS、VEGF、VEGFR-2的蛋白表达升高,同时升高eNOS蛋白的表达（P<0.01）。结论：阿司匹林对LPS诱导的血管内皮细胞炎性损伤的保护作用与调节NOS/NO和VEGF及其受体的动态平衡密切相关。     

细胞穿透肽YARA介导增强型绿色荧光蛋白转导入人结直肠癌细胞的实验研究

目的 构建原核表达载体pET15b-YARA EGFP,表达并纯化出融合蛋白YARA-EGFP,观察其穿透人结直肠癌细胞的情况.方法 用分子克隆技术构建出表达型载体pET15b-YARA-EGFP,在E.coli BL21(DE3)中表达融合蛋白YARA-EGFP,将鉴定和纯化后的融合蛋白加入体外培养的人结直肠癌细胞.结果 得到纯化的融合蛋白YARA-EGFP,能高效地转导入体外培养的人结直肠癌细胞,并表现出剂量和时间依赖性,MTT法检测在其高浓度时仍对细胞无毒性.结论 YARA-EGFP融合蛋白能高效地转导入体外培养的人结直肠癌细胞.     

Honokiol inhibits H(2)O(2)-induced apoptosis in human lens epithelial cells via inhibition of the mitogen-activated protein kinase and Akt pathways

Oxidative stress-induced apoptosis in lens epithelial cells plays an important role in cataract formation, and its prevention may be of therapeutic interest. This study was performed to investigate the protective effect and mechanisms of honokiol on H₂O₂-induced apoptosis in human lens epithelial (HLE) cells. HLE cells (SRA01-04) were pretreated with honokiol at concentrations of 5 μM, 10 μM and 20 μM before 50 μM H₂O₂ treatment. The results demonstrated that pretreatment of honokiol inhibited the activation of caspase-3 and caspase-9 and downregulated the expression of Bcl-2. Mechanistically, honokiol suppressed H₂O₂-induced phosphorylation of ERK1/2, p38 mitogen-activated protein kinase (MAPK), JNK and Akt. Honokiol also inhibited H₂O₂-induced nuclear factor-κB (NF-κB)/p65 phosphorylation and translocation in HLE cells. These results demonstrate that honokiol suppresses H₂O₂-induced HLE cell apoptosis via interference with the MAPKs, Akt and NF-κB signaling, suggesting that honokiol might have a potential effect against cataract formation.     

半边旗中二萜类化合物5F对K562细胞MAPK活性、表达的影响

目的　研究半边旗中二萜类化合物 5F(11α 羟基 15 氧 16 烯 对映贝壳杉烷 19酸 )对K5 6 2细胞MAPK活性、表达的影响。方法　 5F作用K5 6 2细胞 2 4h后 ,以MBP为底物检测MAPK的活性、用蛋白印迹法测定MAPK的表达。结果　半边旗中二萜类化合物 5F作用于K5 6 2细胞使MAPK活性增强 ,表达增多 ,且有量效关系。结论　推测异常激活和表达的MAPK是化合物 5F抗肿瘤的机制之一     

Isorhapontigenin and resveratrol suppress oxLDL-induced proliferation and activation of ERK1/2 mitogen-activated protein kinases of bovine aortic smooth muscle cells

The objective of our study was to compare the inhibitory effect of isorhapontigenin (ISO) and resveratrol, two natural antioxidants, on oxidized low-density lipoprotein (oxLDL)-induced proliferation of bovine aortic smooth muscle cells (BASMCs) and its relation to reactive oxygen species (ROS) generation and extracellular signal-regulated kinase 1/2 activation. The results showed that stimulation of oxLDL (50-150 microg/mL) for 48 hr induced a dose-dependent increase in cell number and incorporation of [3H]thymidine into DNA of BASMCs. Western blot analysis demonstrated that oxLDL (150 microg/mL) stimulated an evident phosphorylation of p42/44 MAP kinases in BASMCs. Incubation of BASMCs with oxLDL induced significant increase in ROS detected by using an oxidant-sensitive fluorescent probe of 2',7'-dichlorofluorescin diacetate. The level of H2O2 in the medium of cultured BASMCs also increased markedly. Preincubation of BASMCs with ISO and resveratrol significantly inhibited oxLDL-induced cell proliferation and incorporation of [3H]thymidine, and the phosphorylation of p42/44 MAP kinases in BASMCs as well. Furthermore, preincubation of BASMCs with ISO and resveratrol attenuated oxLDL-induced increases in ROS and H2O2 levels. The results suggested that oxLDL-induced acute formation of ROS and subsequent activation of redox-sensitive extracellular signal-regulated kinase 1/2 MAPK pathways, which might be important for mitogenic signaling of oxLDL in vascular smooth muscle cells. The inhibitory effect of ISO and resveratrol on oxLDL-induced mitogenesis of BASMCs might be taken through blocking the generation of ROS and activation of the ERKs pathway.     

Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells

Human embryonic and induced pluripotent stem cell-derived hepatocytes (hESC-Hep and hiPSC-Hep) have the potential to provide relevant human in vitro model systems for toxicity testing and drug discovery studies. In this study, the expression and function of important drug metabolizing cytochrome P450 (CYP) enzymes and transporter proteins in hESC-Hep and hiPSC-Hep were compared to cryopreserved human primary hepatocytes (hphep) and HepG2 cells. Overall, CYP activities in hESC-Hep and hiPSC-Hep were much lower than in hphep cultured for 4 h, but CYP1A and 3A activities were comparable to levels in hphep cultured for 48 h (CYP1A: 35% and 26% of 48 h hphep, respectively; CYP3A: 80% and 440% of 48 h hphep, respectively). Importantly, in hESC-Hep and hiPSC-Hep, CYP activities were stable or increasing for at least one week in culture which was in contrast to the rapid loss of CYP activities in cultured hphep between 4 and 48 h after plating. With regard to transporters, in hESC-Hep and hiPSC-Hep, pronounced NTCP activity (17% and 29% of 4 h hphep, respectively) and moderate BSEP activity (6% and 8% of 4 h hphep, respectively) were observed. Analyses of mRNA expression and immunocytochemistry supported the observed CYP and transporter activities and showed expression of additional CYPs and transporters. In conclusion, the stable expression and function of CYPs and transporters in hESC-Hep and hiPSC-Hep for at least one week opens up the possibility to reproducibly perform long term and extensive studies, e.g. chronic toxicity testing, in a stem cell-derived hepatic system.