活性维生素D通过调节糖尿病肾病大鼠巨噬细胞M1及M2表型活化防治足细胞损伤

目的 探讨活性维生素D（VD）能否通过调节肾组织巨噬细胞M1 及M2 表型活化从而防治糖尿病肾病（DN）大鼠足细胞损伤。 方法 利用腹腔注射链脲菌素（STZ）建立糖尿病大鼠模型。将SD 雄性大鼠按随机数字表法分为4 组：对照组1（NC-1 组,n=8）、对照组2（NC-2 组,n=8,对照+骨化三醇0.1 μg·kg^-1·d^-1 灌胃）、DN 组（n=24）、DN+VD 干预组（VD 组,n=24,DN+骨化三醇0.1 μg·kg^-1·d^-1 灌胃）,定期检测血糖、体质量,收集尿标本,分别于干预后8周、14周、18周末处死动物,检测Scr、BUN和尿蛋白变化;PAS染色观察肾脏病理改变;免疫组化法检测肾组织CD68+巨噬细胞浸润数量;Western 印迹法检测nephrin、podocin、CD68 以及M1巨噬细胞特异性标志物诱导型氮氧化物合酶（iNOS）、肿瘤坏死因子α（TNF-α）和M2巨噬细胞特异性标志物CD163、精氨酸酶1（Arg-1）、甘露糖受体（MR）表达。 结果 （1）与两对照组相比,DN 组Scr、BUN、24 h 尿蛋白量及肾小球系膜基质增生程度显著增加（P＜0.05）,podocin、nephrin蛋白表达下降（P＜0.05）。VD干预后能明显改善上述病理现象（均P＜0.05）。（2）与两对照组相比,DN组肾组织CD68⁺巨噬细胞浸润数量明显增加,呈时间依赖性。VD干预后能显著减少CD68⁺巨噬细胞浸润数量（P＜0.05）。（3）进一步确定肾组织巨噬细胞M1、M2活化表型发现,8周、14周、18周末DN组iNOS、TNF-α蛋白表达较对照组显著升高（均P＜0.05）,VD干预后能明显抑制同期DN肾组织iNOS、TNF-α表达（均P＜0.05）;8周、14周末VD组CD163、Arg-1、MR蛋白表达与DN组相比差异无统计学意义（均P＞0.05）,而18周末VD组CD163、Arg-1、MR蛋白表达较DN 组显著升高（均P＜0.05）,CD163/CD68 蛋白表达比例亦显著增加（P＜0.05）。（4）相关分析显示,M1 标志物iNOS 与nephrin、podocin 蛋白表达均呈负相关（r＝-0.707,P＜0.01;r＝-0.712,P＜0.01）;M2标志物CD163与nephrin、podocin蛋白表达均呈正相关（r＝0.627,P＜0.01;r＝0.613,P＜0.01）。 结论 活性维生素D具有调节巨噬细胞表型活化的能力,通过抑制巨噬细胞M1型活化并增强M2型活化,进而发挥足细胞保护作用。     

氯化镧对内毒素/脂多糖诱导巨噬细胞株一氧化氮合酶表达的抑制作用

目的了解氯化镧（LaCl3）对内毒素／脂多糖（LPS）刺激的巨噬细胞诱导型一氧化氮合酶（iNOS）表达的影响，并探讨其机制。方法将小鼠巨噬细胞株RAW264．7分为空白对照组、LaCl、组、LPS组和LaCl3＋LPS组。前3组细胞分别用常规培养液、含2．50μmol／L LaCl3的培养液、含1mg／L LPS的培养液培养24h，LaCl3＋LPS组用含2．5μmol／LLaCl，的培养液培养24h后，换为含1mg／L LPS的培养液培养24h。采用免疫细胞化学染色法检测iNOS在各组细胞中的表达强度；蛋白质印迹法检测iNOS的蛋白表达水平；反转录一PCR测定iNOS的mRNA表达水平；硝酸还原酶法测定各组细胞培养上清液中一氧化氮（NO）含量。结果免疫细胞化学染色结果显示，iNOS主要分布于各组细胞的胞质中，空白对照组和LaCl3组荧光强度极弱；LPS组荧光强度最强，阳性细胞百分率为44．4％，明显高于LaCl3＋LPS组（11．8％，P〈0．05）。LPS组iNOS蛋白及其mRNA表达量和细胞培养上清液中NO含量均高于其余各组（P〈0．05）。结论LaCl3可在mRNA水平和蛋白水平抑制LPS诱导的iNOS过度表达，减少NO生成，提示LaCl3能拮抗LPS诱导的iNOS过度活化。     

Apocynin对缺氧复氧后肾小管上皮细胞MCP-1表达的干预作用

目的：研究缺氧复氧后肾小管上皮细胞单核细胞趋化蛋白-1（MCP-1）的表达及Apocynin对其影响。方法：选用人肾小管上皮细胞（HK-2）建立缺氧复氧损伤模型,设正常对照组、单纯缺氧复氧组、不同浓度（0.05、0.25、0．5mmol／L）Apocynin干预组。流式细胞术检测细胞内氧化应激水平,流式细胞术检测MCP-1蛋白表达、RT-PCR法测定细胞MCP-1 mRNA表达。结果：缺氧复氧后HK-2细胞内氧化应激水平提高,细胞MCP-1蛋白和mRNA表达上调,且随缺氧时间的延长,细胞内氧化应激水平逐渐升高,MCP-1蛋白和mRNA表达逐渐增强;Apocynin呈剂量关系抑制细胞内氧化应激水平,同时呈剂量关系抑制MCP-1蛋白和mRNA表达的上调。结论：缺氧复氧诱导细胞内氧化应激的产生,诱导MCP-1表达上调;Apocynin可以通过抑制细胞内氧化应激抑制MCP-1的上调。     

二甲基乙二酰基甘氨酸对缺氧复氧诱导的肾小管上皮细胞损伤的保护作用及其机制

目的 通过脯氨酸羟化酶抑制剂二甲基乙二酰基甘氨酸（DMOG）稳定缺氧诱导因子1α（HIF-1α）表达,探讨其对缺氧复氧诱导的肾小管上皮细胞（HKC）损伤的保护作用及其机制。 方法 制作无糖缺氧复氧细胞损伤模型,用不同浓度的DMOG预处理,锥虫蓝染色和乳酸脱氢酶（LDH）活性方法检测细胞活力及损伤;Annexin V和PI染色流式细胞仪技术检测细胞凋亡;实时荧光定量PCR方法检测红细胞生成素（EPO）、热休克蛋白70（HSP70）和血红素氧合酶1（HO-1） mRNA的表达;Western印迹法检测HIF-1α、活性caspase-3和Bcl-2蛋白表达。 结果 正常情况下HKC细胞内几乎无HIF-1α蛋白表达,DMOG刺激6 h后HIF-1α蛋白及其靶基因EPO、HSP70和HO-1 mRNA表达均显著上调（均P ＜ 0.01）,且呈浓度依赖性。500 μmol/L或1 mmol/L DMOG预处理可明显改善缺氧复氧诱导的细胞损伤,表现为细胞存活率升高（95.6%±1.8%、96.1%±1.0%比 83.3%±3.1%）;培养上清液中LDH 活性下降;细胞凋亡减少（8.6%±2.7%、6.1%±2.3%比19.2%±4.0%）（均P ＜ 0.05）。另外,细胞内活性caspase-3蛋白表达显著下调,而Bcl-2蛋白表达则显著上调（均P ＜ 0.05）。 结论 DMOG预处理可稳定肾小管上皮细胞内HIF-1α表达,对缺氧复氧诱导的肾小管上皮细胞损伤具有一定保护作用。其机制可能与促进EPO、HSP70和HO-1表达,抑制caspase-3活化,上调Bcl-2表达有关。     

缺氧／复氧后肾小管上皮细胞ICAM-1表达及Apocynin对其干预作用的研究

目的：研究缺氧/复氧后肾小管上皮细胞氧化应激的变化细胞间黏附分子-1（ICAM-1）蛋白和mRNA的表达及NADPH氧化酶抑制剂Apocynin对其的影响.方法：采用肾小管上皮细胞（HK2）建立缺氧（细胞置于37 ℃、95%N2、5% CO2环境中）/复氧（细胞置于37 ℃、95% O2、5% CO2环境中）细胞模型,设正常对照组,缺氧0.5 h、1 h、2 h/复氧24 h组,及不同浓度（0.05 mmol/L、0.25 mmol/L、0.5 mmol/L）Apocynin干预组.流式细胞术检测细胞内氧化应激水平,逆转录-聚合酶链反应（RT-PCR）法测定细胞ICAM-1 mRNA表达,流式细胞术检测ICAM-1蛋白表达.结果：缺氧/复氧后HK2细胞内氧化应激水平提高,ICAM-1 mRNA和蛋白表达增强,随缺氧时间的延长,细胞内氧化应激水平逐渐升高,ICAM-1mRNA和蛋白表达也逐渐增强 Apocynin呈剂量依赖关系抑制细胞内氧化应激和ICAM-1 mRNA、蛋白表达的上调.结论：缺氧/复氧诱导细胞内氧化应激的产生,诱导ICAM-1表达上调 Apocynin可以通过抑制细胞内氧化应激抑制ICAM-1的上调.     

PPARγ激动剂对腹膜透析相关性急性腹膜炎大鼠PPARγ、Toll样受体4表达及STAT1信号活化的影响

目的 观察过氧化物酶体增殖物活化受体γ(PPARγ)激动剂罗格列酮和15脱氧前列腺素J2（15d-PGJ2）对脂多糖（LPS）诱导大鼠腹膜透析相关性急性腹膜炎模型腹膜组织PPARγ、Toll样受体4（TLR4）表达、STAT1活化及腹腔局部炎性反应的影响。 方法 24只雄性SD大鼠随机分成4组，每组6只。对照组：腹腔注入4.25%葡萄糖乳酸盐腹膜透析液（简称腹透液，90 ml/kg）；LPS组：LPS 1 mg/kg腹腔注入4 h后，再注入腹透液；罗格列酮+ LPS组（罗格列酮组）：罗格列酮20 mg&#8226;kg-1&#8226;d-1灌胃预处理3 d，注入LPS及腹透液；15d-PGJ2 + LPS组（15d-PGJ2组）：15d-PGJ2 0.3 mg&#8226;kg-1&#8226;d-1腹腔注入预处理3 d，注入LPS及腹透液。注入腹透液4 h后处死大鼠，留取腹水、壁层及脏层腹膜组织。ELISA法检测腹水中IL-6浓度。常规行腹膜组织Masson染色和腹水白细胞计数。RT-PCR检测腹膜组织PPARγ、TLR4 mRNA的表达；Western印迹法检测腹膜组织PPARγ、TLR4、磷酸化（p）-STAT1、STAT1蛋白的表达。 结果 LPS组大鼠腹水IL-6浓度[268.53（201.87～335.19） ng/L]高于对照组[147.62（130.60～164.64） ng/L）]（P < 0.01）；罗格列酮组大鼠腹水IL-6浓度[110.20（77.60～142.80） ng/L]低于LPS组（P < 0.05）。与对照组比较，LPS组大鼠腹膜组织明显水肿，腹膜组织PPARγ、TLR4 mRNA及蛋白表达均显著增强（P < 0.05）。与LPS组相比，罗格列酮组大鼠腹膜组织水肿明显减轻，PPARγ、TLR4 mRNA表达显著增高（P < 0.05），但其蛋白表达显著减弱（P < 0.05）。15d-PGJ2组大鼠腹膜组织水肿明显减轻，PPARγ mRNA及其蛋白表达均显著减弱（均P < 0.05），TLR4 mRNA表达显著增强（P < 0.01），但其蛋白表达减弱（P < 0.05）。各组间腹水白细胞计数差异无统计学意义。罗格列酮、15d-PGJ2均明显上调LPS诱导的p-STAT1表达（P < 0.01）。 结论 罗格列酮和15d-PGJ2可负性调节LPS诱导的大鼠急性腹膜炎性反应，并对LPS信号通路中相关功能蛋白起一定的调控作用。     

术后引流液外泌体hsa?miR?609在甲状腺乳头状癌发生发展中的作用及其机制探讨

［摘要］ 目的 观察甲状腺乳头状癌（PTC）术后引流液中外泌体microRNA（miRNA）表达变化,探讨其在PTC发生发展中的作用。方法 收集3例PTC患者术后第一天切口引流液,以结节性甲状腺肿术后引流液为对照,采用超速离心法提取引流液中的外泌体,通过透射电镜、粒径分析及标记蛋白检测验证外泌体的特征,提取外泌体中的RNA进行miRNA芯片检测并进行聚类分析。结果 通过囊泡形态、粒径大小及特异性蛋白标记证实术后引流液中存在外泌体,miRNA芯片数据聚类分析结果显示PTC和结节性甲状腺肿患者术后引流液外泌体miRNA谱具有显著差异,其中以hsa?miR?609的表达差异最为显著。生物信息学结果提示预测的hsa?miR?609的靶基因和PTC肿瘤发生紧密相关。结论 术后引流液来源的外泌体中的miRNA表达异常在PTC发生发展中发挥重要调控作用。     

脂氧素A4对脂多糖活化小鼠巨噬细胞NF-κB和TNF-α转化酶的影响

目的 研究脂氧素A4（LXA4）对脂多糖（LPS）活化的小鼠巨噬细胞NF-κB及TNF-α转化酶的影响。方法 小鼠RAW 264．7巨噬细胞株培养于细胞培养板,随机分为空白对照组、LPS处理组（LPS 1μg/ml）和LPS＋LXA4组（LPS 1μg/ml＋LXA4 10^-7mol/L）。酶联免疫吸附法测定上清液TNF-α水平,提取细胞总蛋白,Western blot法测定NF-κB p65、IκB-α及TNF-α转化酶含量,荧光素酶报告质粒测定NF-κB活性。结果 LXA4抑制LPS活化的RAW 264．7巨噬细胞IκB-α降解、NF-κB p65核转位及NF-κB活性,同时抑制TNF-α蛋白表达和上调TNF-α转化酶蛋白表达。结论 LXA4通过抑制NF-κB活性和下调TNF-α转化酶表达而减少LPS活化的RAW264．7巨噬细胞分泌TNF-α。     

硝普钠对内毒素致大鼠急性肺损伤保护作用的机制

目的 研究外源性一氧化氮（NO）供体一硝普钠对内毒索（LPS）致大鼠急性肺损伤（Au）保护作用的机制。方法 32只Wistar大鼠随机分为4组（n=8），假手术组（S组）、内毒素组（LPS组）、HO-1诱导剂氯化高铁血红素预处理组（HM组）和硝普钠治疗组（SNP组）。采用气管内滴入750μg／kg（溶于300ml生理盐水中）LPS建立大鼠ALI模型，HM组在给LPS前12h腹腔注射氯化高铁血红素40μmol／kg，SNP组将LPS与硝普钠（25μg／kg）同时滴入气管。滴人LPS后8h处死大鼠，测定肺组织湿／干重比（W／D）、丙二醛（MDA）含量、诱导型血红素加氧酶（HO-1）、诱导型一氧化氮合酶（iNOS）蛋白表达及支气管肺泡灌洗液（BALF）蛋白浓度，并观察肺组织病理变化。结果 与S组比较，LPS组、HM组、SNP组肺组织iNOS、HO-1蛋白表达均增强，LPS组、HM组肺组织W／D及MDA含量增加，LPS组BALF蛋白浓度增加；与LPS组比较，HM组和SNP组肺组织iNOS蛋白表达减弱，肺组织HO-1蛋白表达增强，肺组织W／D、MDA含量及BALF蛋白浓度降低（P〈0．05或0．01）。SNP及HM组肺组织病理损伤程度明显轻于LPS组。结论 硝普钠可通过诱导HO-1进而抑制iNOS／NO，减轻LPS所致ALI．     

血管内皮细胞EA.hy926对短期低氧的反应性研究

目的观察短期低氧应激对血管内皮细胞的影响。方法制作血管内皮细胞低氧应激模型，采用低细胞毒性的染料AlamarBlue染色法测定常氧和低氧培养条件下的细胞活力，采用流式细胞技术检测细胞周期，采用膜联蛋白V-荧光素／碘化丙锭（Annexin V-fluoreseein／PI）双标记法检测细胞凋亡，应用蛋白免疫印迹方法（Westernblot），检测细胞内缺氧诱导因子和增殖细胞核抗原的表达。结果短期低氧（1～3h）应激可以增强血管内皮细胞活力，细胞周期显示细胞4倍体峰比例增高，随着低氧时间的延长（6～12h），细胞4倍体峰回落至常氧水平。短暂低氧（3h），细胞凋亡比例明显增高。蛋白免疫印迹检测显示，缺氧诱导因子-1α和增殖细胞核抗原在低氧后表达增高，与低氧反应呈时间效应依赖关系。结论短期低氧启动了以增殖和活力增高为特征的血管内皮细胞适应性反应，同时也诱导了以细胞凋亡为特征的细胞损伤性反应。     

The role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells

Objective To investigate the role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells. Methods (1) The supernatant of renal tubular epithelial cells which were cultured in normal glucose control group (5.5 mmol/L D-glucose) or high glucose group (30.0 mmol/L D-glucose) for 48 h were collected and ultracentrifuged to harvest exosomes. Exosomes were identified by transmission electron microscope and Western blotting. (2) Exosomes were labeled with the green lipophilic fluorescent dye PKH67 and cultured with THP-1 macrophage to investigate whether HK2-derived exosomes could be internalized by THP-1 macrophage. Observing the morphology microscopically and detecting the chemotaxis function of THP-1 macrophages in Transwell chamber after co-cultured with exosomes. The expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) in cells and supernatants were separately detected by quantitative Real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), and the expression of p-c-Jun NH2-terminal kinase (p-JNK), mitogen-activated protein kinase p-p38 (p-p38MAPK) and nuclear factor κB p65 (NF-κB p65) in THP-1 macrophages were detected by Western blot. Results (1) Vesicles that harvested by ultracentrifugation ranged in size from 30 nm to 100 nm and expressed exosomal marker CD63, TSG101 but absence of calnexin which is a marker of endoplasmic reticulum, suggesting that the exosomes were not contaminated with cells. (2) Results from laser scanning confocal microscope showed that each group of exosomes can be internalized by THP-1 macrophages. Compared with normal glucose exosomes group, high glucose exosomes had increased the expression of iNOS, TNF-α, IL-1β and MCP-1 in THP-1 macrophages (all P＜0.01), moreover, p-JNK, p-p38 MAPK and NF-κB p65 proteins level also increased significantly (all P＜0.01). Conclusions Exosomes from high glucose-treated HK2 cells can induce THP-1 macrophage activation and functional changes through MAPK/NF-κB pathway.     

TAK1信号通路在高糖诱导骨髓来源巨噬细胞激活中的作用

Objective To investigate the role of transforming growth factor-β activated kinase-1 (TAK1) signaling pathway in the activation of bone marrow derived macrophages (BMDM) induced by high glucose. Methods Purity of mouse BMDM was detected by flow cytometry. The mice macrophages cultured in vitro were stimulated by high glucose and treated with TAK1 specific inhibitor 5Z-7-oxozeaenol. Cells were divided into normal control group (RPMI 1640), osmolality control group (25 mmol/L mannitol), high glucose group (33 mmol/L D-glucose) and inhibitor group (33 mmol/L D-glucose+300 nmol/L 5Z-7-oxozeaenol). Immunocytochemistry and flow cytometry were used to detect macrophage subtype. The expression of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis Factor-α (TNF-α) mRNA were determined by real time PCR. Expressions of p-TAK1, TAK1 binding protein (TAB1), p-JNK, p-p38 MAPK and NF-κB p65 proteins were analyzed by Western blotting. Results The purity of BMDM was about 99.36%. Compared with normal control group, high glucose group had increased percentage of M1 macrophages, increased expression of MCP-1 and TNF-α mRNA (all P＜0.05). Moreover, p-TAK1, TAB1, p-JNK, p-p38 MAPK and NF-κB p65 proteins expression also increased significantly in high glucose group (all P＜0.05). After treatment with inhibitor 5Z-7-oxozeaenol, the effects induced by high glucose were inhibited (P＜0.05). Conclusions High glucose can induce M1 macrophage activation and expression of inflammatory cytokine of BMDM, which can be inhibited 5Z-7-oxozeaenol through inhibiting TAK1/MAPK and TAK1/NF-κB pathway.     

Response of encapsulated rat pancreatic islets to hypoxia

Hypoxia contributes to encapsulated pancreatic islet graft failure. To gain insight into the mechanisms that lead to hypoxia-induced graft failure, encapsulated islet function, vitality, and cell replication were assessed after 2 and 5 days of hypoxic (1% O2) and normoxic (20% O2) culture. The mRNA expression levels of Bcl-2, Bax, inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein 1 (MCP-1) were assessed, as well as the amount of nitrite and MCP-1 in the culture medium. Hypoxia was associated with loss of encapsulated islet function and vitality, but not with an increase in islet cell replication. Loss of vitality was due to necrosis, and only modestly due to apoptosis. Hypoxia was not associated with changes in the Bcl-2/Bax mRNA ratio, but it did increase the expression of iNOS and MCP-1 mRNA. The increased mRNA levels were, however, not associated with elevated concentrations of nitrite nor with elevated levels of MCP-1 protein. The increased iNOS mRNA levels imply a role for NO in the completion of cell death by hypoxia. The increased MCP-1 mRNA levels suggest that encapsulated islets in vivo contribute to their own graft failure by attracting cytokine-producing macrophages. The discrepancy between iNOS mRNA and nitrite is explained by the longer half-life of NO during hypoxia. MCP-1 protein levels are underestimated as a consequence of the lower number of vital cells in combination with a higher proteolytic activity due to necrosis. Thus, strategies to eliminate hypoxia may not only improve islet function and vitality, but may also reduce the attraction of macrophages by encapsulated islets.     

1,25(OH)2D3 promotes M1 macrophage switching to M2 via VDR-PPARγ pathway induced by high glucose

Objective To investigate the effect of 1,25(OH)2D3 on high glucose induced macrophage activation and its underlying signal transduction mechanism. Methods RAW 264.7 cells were used to perform cell culture, the activity of intracellular iNOS was measured. VDR siRNA and PPARγ antagonist pre-treatment with macrophages were done before using 10-8 mol/L1,25(OH)2D3 to intervene high glucose pre-incubated macrophages. M1 markers including iNOS, TNF-α, IL-12, M2 markers including MR, Arg-1, IL-10 and nuclear receptors VDR and PPARγ were separately examined. Results The iNOS activity was increased in a glucose-dose and time dependent manner. Particularly, 25 mmol/L glucose at 24 h gave the maximum response. After being treated with 25 mmol/L glucose for 24 h, not only inflammatory cytokines of TNF-α, IL-12 in the supernatant were increased, but quantitative real-time PCR and Western blotting analysis showed iNOS was also up-regulated (P＜0.05). However, M2 markers, i.e. MR and Arg-1 were significantly decreased (P＜0.05). When in the presence of 1,25(OH)2D3 , the trends were reversed: the markers of M1, including TNF-α, IL-12 and iNOS were obviously reduced (P＜0.05), while M2 markers, IL-10, Arg-1 and MR were increased (P＜0.05). In addition, VDR and PPARγ were also increased (P＜0.05). However, the above effects of 1,25(OH)2D3 were abolished when further inhibited the expression of VDR and PPARγ by VDR siRNA and PPARγ antagonist. Besides, accompanied by VDR, PPARγ was also decreased upon the treatment with VDR siRNA (P＜0.05). Conclusion 1,25(OH)2D3can promote high glucose induced classically activated macrophages (M1) converting to alternatively activated macrophages (M2) and this is achieved through VDR-PPARγ pathway.     

人羊膜间充质干细胞外泌体通过微小RNA-135a促进成纤维细胞迁移实验研究

目的研究人羊膜间充质干细胞外泌体(human amniotic mesenchymal stem cell exosome,hAMSCExo)中的微小RNA-135a(microRNA-135a,miR-135a)对成纤维细胞迁移的作用。方法用外泌体分离试剂盒提取hAMSC-Exo并进行鉴定,划痕实验检测hAMSC-Exo对成纤维细胞迁移的作用。实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)检测过表达miR-135a后hAMSC-Exo中miR-135a基因相对表达量,划痕实验检测过表达和敲减miR-135a后hAMSC-Exo对成纤维细胞迁移的作用,Western blot检测过表达和敲减miR-135a后hAMSC-Exo成纤维细胞迁移相关蛋白[大分子肿瘤抑制因子2(large tumor suppressor 2,LATS2)、E-钙黏着蛋白(E-cadherin)、N-cadherin、α平滑肌肌动蛋白(αsmooth muscle actin,α-SMA)]的相对表达量;均以293T细胞外泌体及hAMSC-Exo作为对照。结果成功获得hAMSC-Exo;划痕实验检测示,hAMSC组促进成纤维细胞迁移能力最强,GW4869(外泌体抑制剂)处理hAMSC组促进成纤维细胞迁移能力减弱。qRT-PCR检测示过表达miR-135a后,hAMSC-Exo中miR-135a基因相对表达量明显增加。划痕实验检测示,过表达miR-135a后,hAMSC-Exo促进成纤维细胞迁移能力增强;而敲减miR-135a后,hAMSC-Exo促进成纤维细胞迁移能力减弱。Western blot检测成纤维细胞迁移相关蛋白显示,与293T细胞外泌体组比较,各hAMSC-Exo处理组均下调Ecadherin、N-cadherin和LATS2表达,上调α-SMA表达;其中过表达miR-135a后,hAMSC-Exo能力增强,敲减miR-135a后,hAMSC-Exo能力较过表达miR-135a组下降。结论hAMSC-Exo中的miR-135a可促进成纤维细胞迁移,抑制E-cadherin、N-cadherin和LATS2表达,促进α-SMA表达。     

Influence of albumin on expression of NLRP3 inflammasome in renal tubular epithelial cells

Objective To investigate the effect of albumin on expression of NLRP3 inflammasome and its downstream cytokines IL-1β and IL-18 in tubular epithelial cells. Methods Thirty mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria were selected, and their renal biopsy samples were stained by PAS and Masson to observe tubular epithelial cells injury and inflammatory cells infiltration. NLRP3, caspase-1, IL-1β and IL-18, as well as different inflammatory cells, were detected by immunohistostaining. In vitro, Western blotting and real-time PCR were employed to detect NLRP3, caspase-1, IL-1β and IL-18 protein and mRNA in HK-2 cells stimulated by bovine serum albumin (BSA) (20 g/L). Results In MsPGN patients with high levels of proteinuria, there were obvious renal tubular epithelial cell injury and inflammatory cells infiltration (all P＜0.05), and the expressions of NLRP3, caspase-1, IL-1β and IL-18 were up-regulated compared to patients with low levels of proteinuria (all P＜0.05). Furthermore, IL-1β and IL-18 expressions were positively correlated with the degree of proteinuria (r=0.836, P＜0.05; r=0.901, P＜0.05). NLRP3, caspase-1, IL-1β and IL-18 protein and mRNA were significantly increased in HK-2 cells stimulated by BSA compared to the control group (all P＜0.05). Conclusions Albumin is able to induce NLRP3 inflammasome activation in tubular epithelial cells, which may be the mechanism of tubulointerstitial injury and inflammation caused by proteinuria.     

miR-155 modulates TNF-α-inhibited osteogenic differentiation by targeting SOCS1 expression

Bone morphogenetic proteins (BMPs) can induce ectopic bone formation, which is negatively regulated by inflammatory cytokines, such as tumor necrosis factor (TNF)-α. Recently, miR-155 has been reported to regulate the transforming growth factor (TGF)-β signaling pathway and inflammatory responses. However, whether and how miR-155 modulates TNF-α-regulated osteogenic differentiation have not been explored. In this study, we demonstrated that miR-155 was involved in TNF-α-mediated inhibition of osteogenic differentiation. Knockdown of miR-155 partially mitigated the inhibition of TNF-α on BMP-2-induced osteogenic differentiation. Bioinformatic analysis identified the candidate target site in the 3' untranslated region (3'UTR) of SOCS1. Knockdown of miR-155 increased SOCS1 protein expression during TNF-α stimulation in MC3T3-E1 cells. And transfection with miR-155 inhibited the wild-type, but not the mutant, 3'UTR of SOCS1-regulated luciferase activity, indicating that SOCS1 is a direct target of miR-155 in osteoblast cells. Furthermore, miR-155 expression could be induced by TNF-α through the JNK pathway. As the result of increased SOCS1 expression, knockdown of miR-155 significantly reduced the JNK/c-Jun activation. In addition, transfection of SOCS1 siRNA or overexpression of SOCS1 coding region could narrow the differences of alkaline phosphatase (ALP) and osteocalcin (OSC) expression between the control and miR-155 inhibitor transfected cells. These data indicated that miR-155 modulates TNF-α-regulated osteogenic differentiation by targeting SOCS1, at least partially through the SAPK/JNK pathway. These findings may provide new insights into understanding the regulatory role of miR-155 in the process of osteogenic differentiation in inflammatory condition.     

血浆外泌体miR-17-5p和miR-21对乳腺癌的诊断价值

目的探讨微小RNA-21（miR-21）和miR-17-5p在乳腺癌患者血浆外泌体中的表达水平及其诊断价值。 方法选取2017年6月至2018年3月于成都市第七人民医院就诊的86例乳腺癌患者为乳腺癌组,选取同期体检健康女性45例为对照组。采用实时定量聚合酶链反应（qRT-PCR）检测miR-21、miR-17-5p在两组血浆外泌体中的表达水平。根据qRT-PCR检测结果将乳腺癌患者分为miR-17-5p高表达组及低表达组,miR-21高表达组及低表达组,比较分析其与乳腺癌患者临床病理参数的关系。采用受试者工作特征曲线（ROC）分析血浆外泌体miR-21、miR-17-5p对乳腺癌的诊断价值。 结果乳腺癌组患者血浆外泌体miR-17-5p表达水平显著低于对照组（P<0.05）,而miR-21表达水平显著高于对照组（P<0.05）;miR-17-5p单独检测时曲线下面积（AUC）为0.677,敏感度为58.14%,特异度为75.56%,截断值为0.72;miR-21单独检测时AUC为0.694,敏感度为59.30%,特异度为77.78%,截断值为1.68;联合检测时敏感度为96.51%,特异度为95.56%,准确性为96.18%;联合检测诊断乳腺癌的敏感度、特异度及准确性均显著高于单项检测（P<0.05）。血浆外泌体miR-17-5p、miR-21表达水平均与TNM分期、分化程度、淋巴结转移、cerbB-2及Ki-67有关（P<0.05）。 结论血浆外泌体低表达的miR-17-5p与高表达的miR-21均可作为诊断乳腺癌的潜在生物学标志物。     

Mechanism of miR-21 in delayed ischemia preconditioning and its protection against subsequent ischemia reperfusion injury in kidney

Objective To investigate the molecular mechanism of protection of ischemia preconditioning on renal ischemia reperfusion injury. Methods Male C57/BL6N mice were randomly divided into two groups: in IR group, 35 min ischemia was induced by occlusion of both renal pedicles followed by 24 h perfusion (I/R). 15 min ischemia was induced 4 days before I/R in IPC group. Blood sample and kidney were collected in IR and IPC group after 24 h perfusion. Serum creatinine (Scr) and histological changes were used to evaluate the renal injury. PHD2 and HIF-1α were evaluated by Western blotting, miR-21 expression was confirmed by real-time PCR. In vitro, hypoxic model was established by 1% O2 in HK-2 cells. Knockdown of miR-21 in hypoxic model was perfermed by locked nucleic acid modified-anti-miR-21 transfection. The levels of miR-21, HIF-1α and PHD2 mRNA were confirmed by real-time PCR. The levels of HIF-1α and PHD2 proteins were tested by Western blotting. Results In vivo, Compared with IR group, the renal function and histological changes were improved in IPC group (P＜0.01). Compared with IR group, the expression of miR-21(P＜0.01) and HIF-1α (P＜0.05) were increased in IPC group, while PHD2 was reduced (P＜0.01). In vitro, hypoxia reduced miR-21. The inhibition of miR-21 could increased the expression of PHD2 (P＜0.05). Conclusions Ischemia preconditioning may exert protection against renal ischemia reperfusion injury by inhibiting PHD2.     

Exosomal circ_HIPK3 reduces apoptosis in H2O2-induced AC16 cardiomyocytes through miR-33a-5p/IRS1 axis

BackgroundExosomal circular RNAs (circRNAs) has been revealed to participate in the processes of cellular angiogenesis, growth and metastasis. Herein, the goal of this work was to investigate the role of exosomal circ_HIPK3 in cardiomyocyte apoptosis.MethodsExosomes were isolated using ultracentrifugation method and observed by transmission electron microscopy (TEM). Western blot was used to detect exosomes markers. The experimental group AC16 cells were exposed to hydrogen peroxide (H2O2). Levels of genes and proteins was detected by qRT-PCR and Western blot. EdU assay, CCK8 assay, flow cytometry, and Western blot were utilized to detect the function of exosomal circ_HIPK3 in proliferation, and apoptosis. The target relationship between miR-33a-5p and circ_HIPK3 or IRS1 (insulin receptor substrate 1).ResultsCirc_HIPK3 was packaged into exosomes and derived from AC16 cells. The expression of circ_HIPK3 was decreased by H2O2 treatment in AC16 cells, which also led to the decrease of circ_HIPK3 in exosomes. Functional analysis showed exosomal circ_HIPK3 promoted AC16 cell proliferation and reduced cell apoptosis under H2O2 treatment. Mechanistically, circ_HIPK3 acted as a sponge of miR-33a-5p to up-regulate the expression of its target IRS1. Functionally, forced expression of miR-33a-5p reversed the reduction of exosomal circ_HIPK3 in apoptosis of H2O2-stimulated AC16 cells. Moreover, miR-33a-5p inhibition contributed to the proliferation of H2O2-stimulated AC16 cells, which was abolished by IRS1 silencing.ConclusionExosomal circ_HIPK3 reduced H2O2-induced AC16 cardiomyocyte apoptosis through miR-33a-5p/IRS1 axis, suggesting a novel insight into the pathology of myocardial infarction.