miR-29a通过调控PTEN表达对脂多糖诱导人肺微血管内皮细胞损伤的保护作用研究*

目的:探讨miR-29a在脂多糖(LPS)诱导人肺微血管内皮细胞(HPMVECs)损伤中的作用及机制。方法:构建LPS损伤HPMVECs模型。RT-qPCR检测miR-29a表达变化;试剂盒测乳酸脱氢酶(LDH)释放量;MTT和流式细胞术分别检测细胞存活率及凋亡率;Western blot测蛋白质表达水平;Microcosm、starBase、Pictar、TargetScan软件预测 miR-29a的可能靶基因,双萤光素酶实验验证miR-29a和PTEN的靶向关系。结果:使用LPS处理HPMVECs,显著降低细胞中miR-29a的表达和细胞存活率,诱导LDH释放量和HPMVECs凋亡率增加,上调细胞中PTEN、Bim蛋白表达,下调p-Akt/Akt、p-FOXO3a/FOXO3a表达 (P<0.05);过表达miR-29a逆转LPS对HPMVECs的损伤作用。萤光素酶报告基因实验证实miR-29a 靶向PTEN,转染miR-29a mimics显著下调PTEN蛋白表达,转染miR-29a inhibitors明显上调PTEN蛋白表达 (P<0.05),但PTEN mRNA表达水平差异均无统计学意义(P>0.05)。结论:过表达miR-29a可能通过抑制PTEN蛋白的表达水平、激活Akt/FOXO3a/Bim信号通路对LPS致HUVECs的损伤发挥保护作用。     

自噬对高糖诱导的人冠状动脉内皮细胞凋亡的保护作用

目的:研究自噬对高糖诱导的人冠状动脉内皮细胞凋亡的影响。方法:将人冠状动脉内皮细胞,分别用常规培养基(正常对照组)、含30 mmol/L D-葡萄糖的高糖培养基(高糖组)、高糖培养基合并雷帕霉素(Rapamycin,RAPA;100 nmol/L)干预(RAPA组)和高糖培养基合并3-甲基腺嘌呤(3-Methyladenine,3-MA,5 mmol/L)干预(3-MA组)培养。利用CCK-8法检测细胞生长活力,使用流式细胞术检测细胞凋亡水平,western blot检测细胞自噬标记蛋白(Beclin1)的表达水平。结果:(1)高糖溶液刺激内皮细胞24 h后,细胞生长活力为正常组的55.0%(P0.01),自噬标记蛋白Beclin1的表达水平明显增加,凋亡水平为正常组的2.0倍;(2)与高糖组相比,RAPA组细胞生长活力明显增加,Beclin1的表达明显升高(P0.01),凋亡水平为高糖组的70.1%;(3)与高糖组相比,3-MA组细胞生长活力明显减少,Beclin1的表达明显降低(P0.01),凋亡水平为高糖组的1.42倍。结论:细胞自噬可能对高糖诱导的人冠状动脉内皮细胞具有凋亡保护作用。     

内质网应激与自噬及其交互作用影响内皮细胞凋亡

内质网应激是普遍存在于真核细胞中的应激-防御机制。在内环境稳态遭到破坏的情况下,未折叠蛋白质反应的3条信号通路,分别通过增强蛋白质折叠能力、减少蛋白质生成和促进内质网相关蛋白质降解等途径缓解细胞内压力。同时,也通过多种分子信号机制调控细胞凋亡。自噬是一种生理性的降解机制。通过形成自噬泡并与溶酶体结合摄取并水解胞内受损细胞器和蛋白质等,清除代谢废物,维持细胞正常功能。自噬缺陷或过度激活均可导致细胞凋亡或非程序性死亡。自噬的程度和细胞内压力水平有关。内质网应激通过未折叠蛋白质反应和Ca²⁺浓度变化及其相关分子信号调控自噬。自噬又可反馈性调节内质网应激反应,二者相互作用,在内皮细胞凋亡过程中发挥重要作用。未来内质网应激和自噬可作为药物靶点为内皮相关性疾病提供诊疗策略。     

饥饿小鼠卵母细胞中自噬和凋亡的电镜研究

小鼠(Mus musculus domesticus)原始卵泡形成在出生后3 d内进行得最剧烈,此时有大量卵母细胞丢失。出生后不久原始卵泡库就建立起来,新生鼠都会经历一段时间饥饿再摄入母乳营养,对出生后的子鼠饥饿处理时,出现了自噬和凋亡的动态变化,自噬和凋亡都可以影响细胞的存活,这很可能与卵母细胞的大量丢失有关。在本项研究中,将对照组子鼠正常母乳喂养,处理组子鼠与母鼠分开,完全不给予母乳。分别收取饥饿1.5 d与2 d子鼠的卵巢制作电镜切片,每组3只子鼠,每只子鼠3张电镜切片,每组共统计9张切片。在电镜下观察其形态变化。通过观察发现,饥饿1.5 d的子鼠卵巢与正常1.5d的子鼠卵巢相比,卵母细胞中的自噬小体数量显著增加。这表明,饥饿处理1.5d促进了卵母细胞的自噬,这可能有助于维持卵母细胞的形态及存活。饥饿处理2 d的子鼠卵巢显示出不同的结果。饥饿2 d的子鼠处于生命的临界阶段,已出现小部分个体死亡。存活子鼠卵巢的电镜形态学观察发现,与正常哺乳2 d的子鼠卵母细胞相比,饥饿2 d子鼠卵母细胞中自噬小体的数量显著减少,并出现了多数卵母细胞凋亡的现象,出现许多凋亡小体。本实验研究结果显示,...     

原代小鼠肺微血管内皮细胞的培养及鉴定

目的建立一种简单高效的小鼠肺微血管内皮细胞原代培养方法。方法选取2～3周ICR小鼠,剪开胸腹腔,取距离肺边缘约1.5mm组织,剪碎成米粒状,置于含有培养液的离心管中,离心洗涤后种瓶进行原代培养。通过细胞形态学观察、细胞Ⅷ因子相关抗原免疫细胞化学染色鉴定所培养的细胞。结果接种24h后,红细胞从贴壁的肺组织块边缘向四周游离;48h后,肺微血管内皮细胞爬出,单个细胞形态为多角形或短梭形,细胞间隙较大,胞核清晰,胞浆丰富;96h后细胞融合,呈典型的单层、铺路石样镶嵌式排列。细胞Ⅷ因子免疫细胞化学染色检测,胞质呈棕红色,表达为阳性,阳性细胞率达98%以上。结论随机组织块法能够成功高效分离培养出原代小鼠肺微血管内皮细胞。     

水解南珠液抑制细胞自噬减轻人微血管内皮细胞氧化损伤

[目的]以人微血管内皮细胞(human microvascular endothelial cell-1,HMEC-1)为研究对象,采用H2O2诱导内皮细胞氧化应激,探讨南珠水解液对HMEC-1细胞氧化损伤的保护作用及机制。[方法]采用生化检验法检测南珠水解液作用后,HMEC-1细胞内超氧化物歧化酶(Superoxide Dismutase,SOD)和谷胱甘肽过氧化物酶(Glutathione Peroxidase,GSH-PX)活性及丙二醛(Malondialdehyde,MDA)含量变化;采用流式细胞仪检测细胞内活性氧(Reactive Oxygen,ROS)含量变化;采用透射电镜观察自噬体;采用免疫荧光实验检测细胞微管相关蛋白轻链3Ⅰ/Ⅱ(Microtubuleassociated protein light chainⅠ/Ⅱ,LC3Ⅰ/Ⅱ)表达变化。[结果]与氧化损伤组相比,不同浓度南珠水解处理组SOD活性显著增加,分别提高8.68%、24.99%和49.43%; GSH-PX活性无显著变化; MDA显著降低,分别降低36.99%、35.82%和60.25%;且给药组细胞内RO...     

肺微血管内皮细胞的原代培养

目的:建立简单高效获取大鼠肺微血管内皮细胞(pulmonary microvascular endothelial cells,PMVECs)的培养方法.方法:(1)组织块贴壁法培养大鼠PMVECs;(2)光镜下观察细胞的形态;(3)扫描电镜和透射电镜分别观察细胞表面及内部的结构特征;(4)免疫荧光法鉴定PMVECs.结果:(1)获得的PMVECs长满后呈典型的铺路石或鹅卵石状;(2)扫描电镜下可见内皮细胞表面存在微绒毛等特殊结构;(3)透射电镜观察可见细胞浆内存在韦伯潘力氏小体(Webel Palade body,WPB);(4)免疫组化结果显示细胞浆中有Ⅷ因子相关抗原存在.结论:改良的组织块法培养肺微血管内皮细胞是一种简单、快捷,有效的方法.     

自噬抑制剂氯喹对急性酒精诱导小鼠肝损伤的影响

目的：探讨自噬抑制剂氯喹（CQ）对急性酒精诱导肝损伤的影响及其作用机制。方法：将雄性C57BL/6小鼠随机分为3组：正常对照组、酒精组、氯喹干预组（n=7）,其中酒精组按4.5 g/kg剂量给予33%（V/V）酒精灌胃。HE和油红O染色检测各组小鼠肝组织脂滴变化;检测肝组织甘油三酯（TG）含量变化;检测血清谷草转氨酶（AST）和谷丙转氨酶（ALT）活性;免疫荧光法检测微管相关蛋白轻链3（LC3）蛋白变化;Western blot法检测LC3蛋白和核蛋白P65表达的变化;ELISA法检测促炎因子TNF-α、IL-6的变化。结果：与对照组比较,酒精组脂滴形成、TG含量、血清AST和ALT活性明显增高。与对照组比较,酒精组LC3-Ⅱ蛋白表达明显增加;与酒精组比较,氯喹干预组使酒精诱导的LC3-Ⅱ蛋白表达增强进一步加剧,使酒精诱导的TG含量、血清AST和ALT活性进一步增高,同时增加了酒精诱导的p65入核及TNFα、IL-6释放。结论：急性酒精能引起小鼠肝脏脂肪变化及炎症,而自噬抑制剂氯喹抑制自噬进程,加剧酒精诱导的肝损伤,说明自噬在酒精诱导肝损伤中可能具有保护效应。     

脂多糖调控对大鼠心脏微血管内皮细胞的转录组分析

目的探索脂多糖(LPS)对大鼠心脏微血管内皮细胞(rCMECs)转录组的调节作用。方法对照组(正常培养rCMECs),LPS组(100 ng/mL LPS处理6 h的rCMECs),每组进行3个生物学重复转录组测序。得到差异基因后,使用实时定量PCR对部分差异基因mRNA的表达进行验证。分别对上调和下调基因进行GO和KEGG富集,并对差异基因进行共表达网络分析。采用独立t检验进行统计学分析。结果LPS处理后,265个基因表达上调,118个基因的表达下调。前10个最显著上调基因为:Mt2a、Cyp7b1、Sod2、Icam1、Ccl2、AC128848.1、Mt1、Cebpd、Serpinb2和Tnfrsf11b。前10个最显著下调基因为:Cavin2、Ankrd1、Edn1、Prss35、Lmod1、Dhrs3、Ttc22、Sema6a、Map2k3和Sema7a。定量PCR的结果表明Mt2a、Sod、Ccl2、Cxcl1、Icam1和Vcaml基因的表达得到了上调(P均<0.01);而Cavin2、Ankrd1、Edn1和Prss35基因表达下调(P均<0.05)。GO和KEGG富集的结果表明,上调基因与内皮细胞对炎性免疫细胞的趋化作用和黏附作用密切相关;而下调基因则是与钙离子信号和G蛋白相关通路以及内皮通透性增加有关。此外,差异基因进行共表达网络分析发现Sod2处于核心位置,提示其可能与LPS诱导的rCMECs的各种变化密切相关。结论LPS调控了rCMECs中大量与炎性免疫细胞进入心肌组织相关基因的表达。     

Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension

Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids.     

Hypoxia-induced apoptosis in endothelial cells and embryonic stem cells

Background: To evaluate the influence of hypoxia and molecular events in endothelial and embryonic stem cells.Materials and Methods: Human umbilical vein endothelial cells (HUVECs) and mouse embryoid body (EB) cells were subjected to hypoxic conditions for different time courses. DNA fragmentation assay, quantification of apoptotic cells by TUNEL assay measured by flowcytometry, and Western blot analysis for the molecular events of apoptosis were performed.Results: DNA fragmentation could be identified under hypoxic conditions in HUVECs and mouse EBs. The DNA fragmentation increased when the hypoxic interval was extended.In situ internucleosomal DNA fragmentation-TUNEL assay also found that the percentages of apoptotic cells increased gradually in HUVECs and mouse EBs when the hypoxic interval was extended. Furthermore, the levels of expression of p53 and Bax both increased in hypoxic conditions.Conclusions: Hypoxia increases both HUVEC and mouse EB apoptosis, which is associated with increase in p53/Bax expression.     

Human and Murine High Endothelial Venule Cells Phagocytose Apoptotic Leukocytes

Apoptotic cell death occurs during normal lymphocyte development and differentiation as well as following lymphocyte exposure to endogenous corticosteroids released during stress, malnutrition, and trauma. Recognition and engulfment of these apoptotic cells is important for the clearance of dying cells before they release potent inflammatory mediators into the vasculature or tissues. Phagocytosis of apoptotic cells is accomplished in part by macrophages. We report for the first time that apoptotic lymphocytes are also phagocytosed by high endothelial venule (HEV) cells. The murine HEV cell line mHEVa rapidly phagocytosed apoptotic lymphoid and myeloid cells with the greatest rate of phagocytosis occurring at 0–6 h. To confirm HEV cell interaction with apoptotic cells, we demonstrated that apoptotic human tonsil lymphocytes were phagocytosed by human tonsil HEV cells in primary cultures. Furthermore, we examined HEV cell phagocytosisin vivo.Mice were treated with a natural corticosterone (4-pregnene-11β,21-diol-3,20-dione) at levels detected during stress or malnutrition (93–180 μg serum cortisol/dl). At 4–12 h posttreatment, apoptotic lymphocytes were present inside vacuoles of HEV cells in axillary lymph node tissue sections, as determined by transmission electron microscopy. These data suggest that, in addition to macrophages, lymph node HEV cells also play a role in the removal of apoptotic lymphocytes. Moreover, since HEV cells are specialized endothelial cells that regulate lymphocyte migration into peripheral lymphoid tissues, they may provide an important checkpoint for clearance of apoptotic lymphocytes within the vasculature, as well as limiting entrance of nonfunctional lymphocytes into the lymph node.     

内源性过氧亚硝基阴离子介导脂多糖导致肺动脉内皮细胞损伤

在培养的牛肺动脉内皮细胞(bovine pulmonary artery endothelial cells,BPAECs)水平上,观察脂多糖(lipopolysaccharide,LPS)对BPAECs诱生过氧亚硝基阴离子(peroxynitrite,ONOO~-)能力及内皮源性ONOO~-在LPS致BPAECs损伤中的作用。结果显示:(1)LPS剂量依赖性地引起BPAECs诱生ONOO~-生成标志物硝基酪氨酸(nitrotyrosine,NT)的荧光强度(即ONOO~-)明显增多,NT阳性细胞数和百分率也明显增多或增高(P<0.05);iNOS选择性抑制剂氨基胍(AG)明显抑制LPS诱生ONOO~-增多(P<0.05),而NT阳性细胞数和百分率分别减少或降低,但无明显差异。(2)在LPS作用下BPAECs培养上清中的MDA含量和LDH活性明显增多和增高,呈现剂量依赖性效应。加AG后MDA含量明显降低(P<0.001),LDH活性呈降低趋势。(3)LPS可诱导BPAECs凋亡明显增多,用EB荧光染色后可见细胞染色质浓集、核变小等凋亡征象。AG可导致LPS引起的BPAECs凋亡明显减少,但仍明显高于溶剂组。LPS可导致BPAECs线粒体呼吸抑制及膜电位下降。上述结果表明,LPS可引起BPAECs生成ONOO~-增多,ONOO~-参与介导LPS所致BPAECs过氧化损伤与细胞凋亡。     

ER stress inhibits neuronal death by promoting autophagy

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress (“preconditioning”) is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro. We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.     

ER stress inhibits neuronal death by promoting autophagy

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress (preconditioning) is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro. We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.     

磷酸三钙磨损颗粒诱导小鼠假体周围骨细胞损伤的作用

目的：研究磷酸三钙（TCP）磨损颗粒是否能诱导小鼠颅骨假体周围骨细胞损伤,并探讨其作用机制。方法：36只雄性ICR小鼠随机分为3组（n=12）：假手术组（Sham）、模型（TCP）组和3-甲基腺嘌呤（3-MA）组,采用TCP磨损颗粒30 mg置于小鼠颅骨中缝骨膜外缝合皮肤构建小鼠颅骨溶解模型。3-MA处理组小鼠于术后第2天颅顶皮下注射自噬的特异性抑制剂3-MA （1.0 mg/kg）,2 d 1次,2周后处死动物取血清和颅骨。Micro-CT分析各组小鼠颅骨骨密度（BMD）、骨体积分数（BVF）和骨吸收孔（porosity）数;HE染色和流式细胞术检测各组假体周围骨细胞活性及凋亡情况;ELISA法检测各组血清中骨细胞特征蛋白牙本质基质蛋白（DMP-1）和骨硬化蛋白（SOST）水平;Western blot法检测各组假体周围骨细胞中DMP-1、SOST和自噬关键分子Beclin-1及微管相关蛋白1轻链3（LC-3）等蛋白的表达。结果：与Sham组比较,TCP组假体周围骨细胞活性明显降低,骨细胞死亡及凋亡显著增加（P<0.05）,血清SOST水平及其蛋白表达明显增加,而血清DMP-1水平及其蛋白表达显著减少（P<0.05）,Beclin-1表达增加,LC-3I向LC-3Ⅱ转换明显增加;与TCP组比较,3-MA组假体周围骨细胞损伤明显加重,骨细胞凋亡显著增加（P<0.05）。结论：TCP磨损颗粒可通过激活凋亡和自噬而诱导假体周围骨细胞损伤,促进假体周围骨溶解和关节无菌性松动。