低氧促进P19细胞向多巴胺能神经元分化

目的观察低氧对P19细胞神经分化的影响,针对其向多巴胺能神经元分化的现象及机制进行探讨。方法实验分为常氧组(20%O2)和低氧组(3%O2,每天低氧10 m in)。对诱导分化的神经元采用免疫细胞化学染色方法鉴定。用流式细胞术及W estern b lot检测多巴胺能神经元,高效液相色谱法测定分泌的多巴胺。用RT-PCR技术检测低氧诱导因子HIF-1αmRNA水平。结果①在分化的P19细胞中,低氧组神经元含量高于常氧组(P<0.05),低氧组多巴胺能神经元含量及所分泌的多巴胺显著高于常氧组(P<0.001);②低氧组诱导期HIF-1αmRNA表达水平明显高于常氧组。结论低氧可以促进P19细胞的神经分化,尤其促进P19细胞向多巴胺能神经元分化,HIF-1α可能在其中起了一定作用。     

P19细胞心肌体外分化诱导条件的优化

目的分别观察P19细胞心肌分化诱导中的几种影响因素,优化P19细胞心肌分化的诱导条件。方法使用悬滴法或96孔板悬浮培养法制备细胞聚集体(aggregates),二甲基亚砜(DMSO)为诱导剂诱导P19细胞的心肌分化。普通光学显微镜下观察跳动的心肌细胞的产生来确定诱导是否成功,免疫荧光染色确认心肌特异性蛋白Tro-ponin T的表达,RT-PCR方法检测分化诱导后心肌细胞标志基因的表达情况。结果使用96孔板悬浮培养法诱导细胞形成聚集体后转入贴壁培养阶段时细胞聚集体贴壁状态优于传统的悬滴法。DMSO的加入可以提高细胞形成聚集体后贴壁时细胞聚集体的完整性。1×107~2×108L-1的接种浓度下心肌细胞分化诱导效率较高。在可观察到跳动的心肌细胞之前即可检测到Troponin T的表达。结论通过对细胞形成聚集体的诱导方法、诱导剂、接种浓度等因素的优化可以提高P19细胞的心肌分化效率,为开发高效心肌分化诱导法提供了条件。     

Eosinophil cationic protein enhances cardiomyocyte differentiation of P19CL6 embryonal carcinoma cells by stimulating the FGF receptor signaling pathway

We investigated the functional role of eosinophil cationic protein (ECP) in regulating cardiomyogenesis using mouse P19CL6 embryonic carcinoma cells. ECP was confirmed to accelerate the cardiomyocyte differentiation of P19CL6 cells by enhancing the rate and area size of beating of cardiomyocyte and by facilitating the expression of cardiomyocyte-specific genes, such as GATA4 and α-MHC. Since cardiomyocyte differentiation in vivo is considered to follow mesoderm induction, the induction of Brachyury, a marker of mesoderm, was assessed. Brachyury expression was found to be enhanced after the addition of ECP. This enhancement was due to the stimulation of extracellular signal-regulated kinase (ERK)1/2 phosphorylation by ECP. In this context, treatment with SU5402, an inhibitor of fibroblast growth factor (FGF) receptor 1, suppressed Brachyury expression, phosphorylation of ERK1/2, and cardiomyocyte differentiation induced by ECP. We concluded that ECP might induce mesoderm differentiation through FGF signaling pathway and enhance subsequent cardiomyocyte differentiation in concert with dimethyl sulfoxide in P19CL6 cells. ECP may be a novel factor for cardiomyocyte differentiation, which should be very useful to prepare adequate numbers of cardiomyocytes for therapeutic cell transplantation.     

过表达P75神经生长因子受体负向调控骨髓间充质干细胞的成骨分化

文题释义： P75神经生长因子受体：即P75^NTR,神经生长因子的受体之一,以三聚体和单体的形式镶嵌于细胞表面,主要包含胞外区、跨膜区以及胞内区3个结构区域。低聚状态决定了其对细胞的主要调节功能,与配体结合后通过改变构象来进行信号传导,进而发挥调节细胞的作用。 成骨分化：多向分化干细胞向单能成骨细胞转变,并最终向骨组织发展的一系列生理过程称为成骨分化,对调节骨形成和骨再生具有重要意义,其成骨分化过程中,碱性磷酸酶的活力逐渐增加,细胞内或者胞外基质的钙等矿物质沉积,形成钙结节,骨钙素及Ⅰ型胶原蛋白表达增加。背景：P75^NTR广泛表达于神经组织及细胞中,并发挥着促进或抑制分化的双重作用;同时在骨折不愈合局部组织中也发现了P75^NTR存在着过表达,因此研究P75^NTR对骨髓间充质干细胞成骨分化的作用具有重要的意义,为临床上提高骨折不愈合修复的疗效提供重要靶点。 目的：观察P75^NTR过表达对大鼠骨髓间充质干细胞体外诱导成骨分化的影响。 方法：选取SD大鼠双侧股骨,用全骨髓分离贴壁法提取大鼠骨髓间充质干细胞并进行体外传代培养;构建表达EGFP的P75^NTR过表达质粒GV358-P75^NTR,并用空慢病毒载体包装收集P75^NTR过表达慢病毒载体;选取体外培养至原代10 d的大鼠骨髓间充质干细胞,消化后种板,加入P75^NTR过表达慢病毒及相关感染试剂进行感染实验,感染7 d后用荧光显微镜观察绿色荧光蛋白的表达,Western blot检测P75^NTR蛋白的过表达情况;感染后用常规培养基培养7 d,更换成骨诱导分化培养基培养,诱导培养后第7,10,14天用酶标法定量检测碱性磷酸酶活力,诱导培养后第7,14天用茜素红染色观察矿化结节形成情况。 结果与结论：①P75^NTR过表达慢病毒感染骨髓间充质干细胞后能表达出绿色荧光蛋白(感染效率约90%),且P75^NTR蛋白表达量明显增多,与阴性病毒对照组比较差异有显著性意义(P < 0.05),过表达P75^NTR细胞模型构建成功;②与阴性病毒对照组及未转染组相比,P75^NTR过表达组细胞诱导分化后相应时间点的碱性磷酸酶活力明显降低,矿化结节形成减少,细胞聚集分布减弱,差异有显著性意义(P < 0.05);③结果表明,P75^NTR过表达负向调控了大鼠骨髓间充质干细胞的成骨分化。局部组织中P75^NTR过表达抑制周围骨髓间充质干细胞成骨分化可能是骨缺损或骨折不愈合的重要因素。 ORCID: 0000-0001-6923-3177(朱伦井) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Scavenger receptors as regulators of natural antibody responses and B cell activation in autoimmunity

Innate immune activation is crucial in defense against invading pathogens, including recognition by pattern recognition receptors, such as scavenger receptors. The scavenger receptor family was originally defined by their ability to bind oxidized LDL and thus the majority of research on this set of receptors has been done in association with cardiovascular disease. However, these receptors also bind an array of other modified self and foreign ligands and have for this reason the ability to regulate the immune response, including B cell activation. In this respect, increasing evidence suggests that these receptors are involved in autoimmunity and might provide a link between autoimmune disease and atherosclerosis. In this review, we will summarize how scavenger receptors can regulate activation of B cells both through their expression on this cell type but also by functions mediated by expression on cells interacting with B cells. Recent evidence of scavenger receptor function reveals how the transition from natural and polyreactive antibody responses towards potentially pathogenic B cell activation occurs. This translates to a new role for scavenger receptors in atherosclerosis and autoimmune disease, such as systemic lupus erythematosus.