低氧环境可体外促进人诱导多能干细胞分化为拟胚体

目的 探讨人诱导多能干细胞向拟胚体分化过程中,生理性低氧环境在拟胚体悬浮和贴壁阶段的影响及其相关机制。方法 本研究中拟胚体悬浮阶段分为悬浮常氧组（21% O2）和悬浮低氧组（5% O2）,贴壁阶段分为贴壁常氧组（21% O2）、贴壁低氧组（5% O2）、贴壁低氧组＋HIF-1α抑制剂Echinomycin。首先采用免疫荧光法鉴定人诱导多能干细胞的多能性,人诱导多能干细胞消化后分别在21%氧气和5%氧气条件下悬浮培养5 d,利用显微镜观察悬浮阶段拟胚体的形成和形态变化,5 d后检测拟胚体中三胚层标记基因的表达情况。接着分别取等量的拟胚体接种到明胶包被的培养皿,继续在21%和5%氧气条件下培养2 d,观察贴壁阶段拟胚体的粘附性能和粘附后向外周扩增速度的差异,检测低氧信号通路相关的HIF-1α、β-catenin和VEGFA的表达情况,随后在5%氧气条件下使用HIF-1α特异性抑制剂Echinomycin后,再次检测低氧信号通路相关的HIF-1α、β-catenin和VEGFA的表达情况。结果 常氧组和低氧组的拟胚体均具有三胚层分化的潜力。与常氧培养组相比,低氧组悬浮拟胚体周围的凋亡碎片明显减少,且较早出现囊性拟胚体,获得的拟胚体大小更加均匀一致。此外,低氧组的拟胚体贴壁数量明显高于常氧组（P<0.05）,拟胚体贴壁后向外生长的速度明显快于常氧组（P<0.05）。低氧组HIF-1α的mRNA未显著上调（P>0.05）,而β-catenin和VEGFA的mRNA显著上调（P<0.05）;在蛋白质水平,低氧信号通路相关的HIF-1α、β-catenin和VEGFA的表达均上调（P<0.05）。低氧组使用HIF-1α特异性抑制剂Echinomycin后,低氧信号通路相关的HIF-1α、β-catenin和VEGFA的蛋白质表达均下调（P<0.05）。结论低氧条件不影响拟胚体的三胚层分化潜力。生理性低氧可以促进悬浮拟胚体的形成和成熟,并且能增强拟胚体的贴壁和贴壁后增殖性能,初步证实通过激活HIF-1α/β-catenin/VEGFA信号通路,提升人诱导多能干细胞的分化能力。     

小檗碱通过激活 Nrf2-HO-1/GPX4 通路抑制小鼠海马神经元HT22细胞的铁死亡

目的 探讨小檗碱对于Erastin诱导小鼠海马神经元HT22细胞的铁死亡的保护作用及其可能机制。方法 以HT22小鼠海马神经元细胞为研究对象,分为对照组、Erastin模型组、Erastin+30 μmol/L BBR组、Erastin+60 μmol/L BBR组。采用CCK-8法、特异性 Fe2+ 荧光探针、荧光染料（DAPI）检测和荧光探针（H2DCFH-DA）检测各实验组细胞的增殖情况、活性铁水平、细胞凋亡和活性氧（ROS）变化。 RT-qPCR和Western blot分别检测各实验组细胞的Nrf2、HO-1、GPX4 mRNA和蛋白表达情况。以60 μmol BBR的最适浓度来进一步探究其作用机制,分为对照组、Erastin模型组、Erastin+60 μmol/L BBR组、Erastin+60 μmol/LBBR+2 μmol Nrf2抑制剂 ML385组。通过使用荧光探针和Western blot检测Nrf2抑制剂（ML385）作用后的活性铁的水平、活性氧含量以及Nrf2、HO-1、GPX4蛋白的表达来验证小檗碱调节的Nrf2-HO-1/GPX4通路对Erastin处理的HT22细胞的保护作用。结果 0.5 μmol/L Erastin作用于HT22细胞8 h,细胞存活率与对照组相比显著被抑制（P<0.05）;同时细胞凋亡、ROS以及活性铁含量增加（P<0.05）。与Erastin组比较,Erastin+30 μmol/L BBR组和Erastin+60 μmol/L BBR组的细胞存活率明显升高（P<0.05）,同时显著降低细胞凋亡、ROS以及活性铁含量（P<0.05）。小檗碱增加 HT22细胞中Nrf2、HO-1、GPX4基因及蛋白的 表达量（P<0.05）。加入Nrf2抑制剂ML385后,Nrf2-HO-1/GPX4通路被抑制,并且ROS以及活性铁含量升高（P<0.05）。结论 Erastin诱导HT22细胞发生铁死亡,小檗碱抑制Erastin诱导的铁死亡,可能机制是激活了Nrf2-HO-1/GPX4通路。     

大鼠永久性脑缺血后脑内Nestin的表达变化

目的 研究永久性脑缺血大鼠脑内巢蛋白（neuro epithelial stem cell prote,Nestin）的表达变化,探讨脑缺血后神经干细胞的增生、迁移和分化规律。方法 采用微创开颅法复制大鼠大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）模型,分为正常对照组、假手术组和脑缺血组;假手术组和脑缺血组又分为术后1d、2d、3d、1周、2周、3周和4周共7个亚组,抗Nestin抗体和抗Nestin/胶纸原纤维酸性蛋白（glial fibrillary acidic protein,GFAP）抗体分别进行免疫组化单标和免疫荧光双标染色。结果 正常对照组和假手术组仅在部分血管内皮、侧脑室室管膜下区（subventricular zone,SVZ）及第三脑室周围见极少量的Nestin阳性表达,阳性细胞呈椭圆形或不规则形,体积小,突起短少。脑缺血后Nestin阳性细胞明显增多,体积变大,呈多突起的星形,且大部分阳性细胞与星形胶质细胞的标志物GFAP共表达;增生的阳性细胞从缺血侧SVZ梯度式迁往梗死灶边缘,于缺血后3d达到增生高峰,之后增生能力逐渐下降,缺血后2周,缺血侧SVZ的Nestin阳性细胞数及吸光度值已回落到正常水平;缺血4周后,梗死灶周围的阳性细胞也基本消失。结论 大鼠永久性脑缺血后激活了SVZ的Nestin阳性细胞一过性增生、迁往梗死灶周围并分化为星形胶质细胞,可能对缺血损伤后的脑组织起到修复作用。     

Short-term outcomes of a simple and effective approach to aortic root and arch repair in acute type A aortic dissection

Objective

To evaluate short-term outcomes following direct aortic root and arch repair in patients with acute type A aortic dissection (ATAAD) without technical adjuncts.

Deferoxamine preconditioning activated hypoxia-inducible factor-1α and MyD88-dependent Toll-like receptor 4 signaling in intestinal stem cells

Background/Purpose

Toll-like receptors (TLRs) are important regulators of innate immunity, and TLR4 pathway can regulate the survival, migration, and differentiation of stem cells, including intestinal stem cells (ISCs). Deferoxamine (DFO), a hypoxia-mimic compound, can activate the proliferation of ISCs. In this study, we investigated the response of TLR4 signaling to DFO-induced hypoxia in cultured ISCs in vitro.

Donor mesenchymal stem cell linetics after transamniotic stem cell therapy (TRASCET) for experimental spina bifida

Purpose

We sought to examine donor mesenchymal stem cell (MSC) kinetics after transamniotic stem cell therapy (TRASCET) in experimental spina bifida.

Oestradiol‐17β is a local factor inducing the early stage of spermatogenesis in mouse testes

This study was undertaken to elucidate the effect of testicular oestradiol‐17β (E₂) on spermatogenesis. Spermatogonial development and spermatogenic gene expression in testicular germ cells were investigated using an in vitro culture system supplemented with E₂. E₂ stimulated spermatocytogenic development of cultured testicular germ cells regardless of the addition of follicle‐stimulating hormone and testosterone to the culture medium. E₂ also induced the expression of genes encoding synaptonemal complex protein 1 and protamine 1, proteins required for spermatogenesis. In conclusion, the results of this study suggest that E₂ is a spermatocytogenic factor that acts via the stimulation of spermatogenic gene expression.     

Screening of HLA sensitization during acute burn care

Background

Since the inception of clinical VCA almost two decades ago, burn victims have been identified as immunologically complex patients owing to preformed HLA antibodies. However, it remains unclear whether the detected HLA antibodies are the result of former alloantigenic events or if their de novo formation occurs during primary burn care.