胎肺细胞肾包膜下种植模型——胎肺发育研究之新体内模型

肺是哺乳动物重要的呼吸器官,其发育的大部分过程发生于胚胎阶段,但由于研究手段的限制,对胎肺特别是后期胎肺发生机制的认识还十分有限。本文利用肾包膜下种植的方法建立了胎肺细胞肾包膜下种植模型。模型中上皮发育历经假腺体期、小管期和肺泡前体期等正常胎肺上皮组织发育的所有分化阶段,同时间充质形成广泛的毛细血管网络,与胎肺在子宫中的发育过程一致。更重要的是,消化处理后的单个胎肺细胞可有效地吸收反义寡核苷酸,并在种植组织中产生相应的表型效应。模型的建立为胎肺发生机制研究提供了一条新的途径。     

胎肺细胞肾包膜下种植模型--胎肺发育研究之新体内模型

肺是哺乳动物重要的呼吸器官,其发育的大部分过程发生于胚胎阶段,但由于研究手段的限制,对胎肺特别是后期胎肺发生机制的认识还十分有限.本文利用肾包膜下种植的方法建立了胎肺细胞肾包膜下种植模型.模型中上皮发育历经假腺体期、小管期和肺泡前体期等正常胎肺上皮组织发育的所有分化阶段,同时间充质形成广泛的毛细血管网络,与胎肺在子宫中的发育过程一致.更重要的是,消化处理后的单个胎肺细胞可有效地吸收反义寡核苷酸,并在种植组织中产生相应的表型效应.模型的建立为胎肺发生机制研究提供了一条新的途径.     

小鼠胎肝间充质干细胞在缺血脑组织中迁移机制的研究

目的：探讨小鼠胎肝间充质干细胞（flMSCs）在缺血脑组织中迁移的机制。方法：分离和培养小鼠flMSCs,制备小鼠脑缺血再灌注模型,RT-PCR方法检测小鼠flMSCs表达的趋化因子受体及其唯一配体基质细胞来源因子1α（SDF-1α）在缺血损伤脑组织中的n1RNA表达;Westernblot检测SDF-1α蛋白在缺血损伤脑组织中的表达;免疫组织化学检测SDF-1α在缺血损伤脑组织中的表达和分布;Boydenchamber法进行SDF-1α诱导flMSCs迁移的体外实验。结果：flMSCs经RT-PCR检测表达趋化因子受体CR1,CR3,CXCR1,CXCR2,CXCR3,CXCR4。脑缺血损伤侧脑组织SDF-1αmRNA表达显著增高,与正常脑组织SDF-1αmRNA比,具有显著差异（P〈0．01）,Westernblot检测显示缺血侧脑组织SDF-1α蛋白表达量在12、24、48h分别为0．35±0．05,0．88±0．04,0．74±0．07,与正常脑组织SDF-1α蛋白（0．22±0．04）比,差异有显著性（P〈0．01）。免疫组织化学检测显示,缺血损伤后24h,缺血侧脑皮质,海马等缺血边缘区SDF-1α表达显著增高,缺血对侧及正常脑组织未见明显SDF-1α表达。体外迁移实验显示SDF—1α体外可以趋化flMSCs发生迁移,CXCR4阻断抗体可以阻断SDF—1α诱导flMSCs发生的迁移。结论：SDF-1α可以诱导flMSCs发生迁移,趋化因子受体CXCR4及其配体SDF-1α的相互作用是flMSCs在缺血损伤脑组织中迁移的机制之一.     

骨形态发生蛋白在脂肪生成中的作用

骨形态发生蛋白（bone morphogenesis proteins, BMP）是一类多功能生长因子,除BMP1外都属于转化生长因子β（transforming growth factor beta, TGFβ）超家族的成员. 近年来,越来越多的研究表明,BMP在脂肪生成过程中也起着重要的作用. 本文综述了BMP在诱导间充质干细胞（mesenchymal stem cells, MSC）、脂肪前体细胞系和胚胎干细胞（embryonic stem cells, ESC）生成脂肪细胞的过程中的作用及信号通路方面的研究进展.这些结果将有助于了解不同部位脂肪沉积的调控机制以及一些脂肪过多疾病（如肥胖症）的产生原因     

骨髓间充质干细胞定向分化的经典WNT机制

骨髓间充质干细胞的定向分化一直是干细胞研究的重点,在其分化过程中有多条信号通路参与和调节。目前,Wnt通路在骨髓间充质干细胞定向分化过程中的作用是国外的研究热点。研究发现经典Wnt通路的激活与骨髓间充质干细胞的定向分化高度相关,故将其近年来的研究综述如下,从而为骨质疏松等疾病的治疗以及骨组织工程的发展提供必要的参考依据。     

小鼠骨髓间充质干细胞通过miR-130b调控上皮钠通道的机制研究

该研究旨在探讨小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)通过miR-130b对上皮钠通道(epithelial sodium channel,ENaC)的影响。将分离与培养的小鼠BMSCs接种到Transwell小室中,然后与H441细胞进行共培养。利用CCK-8试剂盒检测BMSCs对H441细胞生存能力的影响;采用Western blot技术检测BMSCs对共培养的H441细胞中γ-ENaC蛋白水平的影响;qRT-PCR技术检测与BMSCs共培养的H441细胞中miR-130b表达情况,然后将此microRNA转染到普通培养的H441细胞中,在蛋白水平进一步验证其对H441细胞中γ-ENaC的影响。实验结果表明,BMSCs能够增强H441细胞的生存能力;同时BMSCs能分别增加共培养的H441细胞中γ-ENaC的蛋白水平以及miR-130b的转录水平;Western blot实验进一步证实,miR-130b转染至H441细胞后能够增加其γ-ENaC的蛋白表达。由此我们推测,BMSCs能够增强H441细胞的生存能力并且可能通过miR-130b发挥其对γ-ENaC的蛋白水平调控作用。     

Notch 通路在大鼠肝纤维化发生发展中作用的初探

目的:研究Notch通路在肝纤维发生发展中作用及可能的分子机制。方法:Wistar大鼠40只随机分为正常对照组与病理模型组,病理模型组皮下注射四氯化碳制备肝纤维化模型。8周后将大鼠处死,取肝组织行病理HE染色评价肝纤维化程度并采用免疫组织化学法检测Notch-1蛋白、E-cadherin蛋白与TGF-β1蛋白的表达。结果:肝组织病理HE染色示肝纤维化大鼠肝脏肝细胞坏死、再生明显,胶原纤维沉积明显增加,肝实质结构紊乱。与正常对照组相比,病理模型组notch-1与TGF-β1蛋白表达明显增加,而E-cadherin蛋白的表达明显下降(P<0.01)。结论:Notch通路在大鼠肝纤维化发生发展中可能起重要作用。     

斑马鱼胚胎背腹轴建立的分子机制

脊椎动物胚胎发育起始于体轴的建立,是胚胎早期发育过程中最重要的事件之一。Wnt、BMP、Nodal和FGF等多个信号通路协同调控细胞分化和细胞运动,促进胚胎胚层的形成和空间上的分离,调控胚胎背腹轴、前后轴和左右轴线的分化,为胚胎进一步发育勾勒出蓝图。本文主要综述斑马鱼胚胎背腹轴建立的分子机制,包括背部组织中心简介;母源Wnt/β-catenin信号调控背部组织中心形成的分子机制;BMP信号调控背腹轴建立的分子机制。     

间充质干细胞向成骨细胞分化的信号通路

间充质干细胞具有向成骨细胞分化的潜能,可体外分离、培养和扩增,是骨组织工程中理想的种子细胞。近年的研究表明间充质干细胞的成骨分化受到多种信号通路的调控,现就其中研究较为深入的MAPK和Notch通路的情况作一简要综述。     

心肌再生相关分子的调控机制

心脏是脊椎动物的中心器官,其适当大小及功能在整个生命周期都是至关重要的。由于心肌损伤造成的心肌梗死、心力衰竭等疾病在全世界范围内的发病率和死亡率逐年上升,目前依然没有找到好的治疗方法。已经发现在新生哺乳动物以及低等脊椎动物中存在多种进化保守的心脏再生机制,然而不幸的是,成年哺乳动物的心脏再生能力极其有限。近年来人们对心肌再生的研究越来越多,有证据表明成年哺乳动物可以产生新的心肌细胞。了解心脏再生的能力,并且掌握其中的原理是心血管方向研究的重要目标。本文主要综述了心肌再生相关分子及信号通路,如转录因子GATA4、微小RNA(microRNA)、Hippo信号通路、ERBB2和Notch通路以及一些炎症因子等发挥的调控作用及其机制。     

Mouse Embryonic Lung Culture,A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.Download video file.^{(87M, mp4)}     

Branching Morphogenesis of Mouse Embryonic Submandibular Epithelia Cultured under Three Different Conditions

To investigate how the mesenchyme interacts with the epithelium, we employed three different culture systems: System A, in which intact submandibular gland rudiments at the mid 13-day stage were cultured on Millipore filters; System B, in which the 13-day epithelium and mesenchyme were separated once with dispase, recombined again, and cultured on the filter; System C, in which the separated 13-day epithelium was clotted with Matrigel and cultured with the mesenchyme across the filter or in the presence of EGF instead of the mesenchyme. In Systems A and B, 13-day epithelia expanded and produced similar lobules with narrow clefts and stalk. When the 13-day epithelium was cultured in System C under the influence of the mesenchyme, it formed rather oval lobules with stalk that were superficially similar to those in System A, but narrow clefts, as seen in the intact early 13-day gland, were rarely found in System C. Furthermore, no long stalk formation was observed when EGF was introduced in place of the mesenchyme. A bacterial collagenase from Clostridium histolyticum gave a considerable inhibition of branching of the 13-day epithelium in Systems A and B, but no significant inhibition was observed in System C when the mesenchyme or EGF was employed as the source of diffusible factor(s). In contrast, although the 13-day epithelium was significantly resistant to the action of heparitinase I from Flavobacterium heparinum in Systems A and B, the enzyme almost completely inhibited the expansion and branching of the epithelium in System C. Judging from these observations, we conclude that the mechanisms of lobular formation in Systems A and B are not the same as those in System C, where the epithelium is clotted with basement membrane matrix components during tissue culture.     

维持胚胎干细胞自我更新状态的分子信号途径

胚胎干细胞是从胚胎植入前期胚泡内细胞团分离的细胞,可以长久维持对称性自我更新的未分化状态。多种胞内外细胞因子介导的信号途径参与这种状态的调节。现对胚胎干细胞自我更新途径分子机制进行综述,并提出有待进一步阐明的相关问题。     

TGF-β1 Inhibits Growth and Branching Morphogenesis In Embryonic Mouse Submandibular and Sublingual Glands in Vitro

Members of the TGF-β superfamily of polypeptides are key regulators in developmental processes. Several studies have shown that expression of TGF-β mRNA and protein are developmentally regulated and that both are prominently expressed in tissues undergoing epithelial-mesenchymal interactions such as branching morphogenesis. It has been shown that TGF-β1 protein is present in E 14 mouse submandibular glands at a time when branching is already establihsed. Here we demonstrate by RT-PCR and immunofluorescence that both TGF-β1 mRNA and protein are present in E 13 submandibular and sublingual glands at a time when branching is being initiated. Addition of TGF-β1 to E 13 rudiments resulted in reductions in organ size and inhibition of branching. Sensitivity to TGF-β1 depended on the developmental stage of the rudiments (early or late E 13) and the dose of growth factor used. TGF-β1 Also caused epithelial abnormalities, notably treated organs had elongated ducts. The effects were most pronounced in the sublingual gland. Taken together these results suggest a regulatory role for endogenous TGF-β1 in the growth and morphogenesis of mouse salivary glands.     

Fetal Calcium Regulates Branching Morphogenesis in the Developing Human and Mouse Lung: Involvement of Voltage-Gated Calcium Channels

Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9–17 of human gestation, embryonic days (E)11.5–16.5 in mouse) in a hypercalcaemic environment (∼1.7 in the fetus vs. ∼1.1–1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca²⁺ channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (Ca_V1.2 and Ca_V1.3), P/Q type (Ca_V2.1), N-type (Ca_V2.2), R-type (Ca_V2.3), and T-type (Ca_V3.2 and Ca_V3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Ca_v1.2 and Ca_v1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Ca_v2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Ca_v2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC₂(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match growth and distension within the developing lung.     

Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/β-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.     

维持胚胎干细胞自我更新分子机制的研究进展

胚胎干细胞通过特殊内源性分子的表达,以及微环境中多种细胞因子和胞外基质的刺激,构成信号网络,共同调控自我更新.近年来,通过对Oct3/4、Nanog等胚胎干细胞特殊分子标记,以及LIF-STAT3,Wnt-β-连环素,BMP-Id等信号通路的研究,探讨了胚胎干细胞自我更新信号网络的分子机制.维持自我更新的关键在于胚胎干细胞生长微环境中的各种细胞因子和胞外基质的含量,以及细胞内源性特异分子表达量之间的平衡.     

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1''-dioctadecyl-3,3,3'',3''-tetramethylindocarbocyanine perchlorate (DiI)¹. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching² (Fig. 1).The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)³. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself⁴ (Fig. 2), however, other options have been discussed as well⁵.This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.     

The ERK-1/2 Signaling Pathway Is Involved in the Stimulation of Branching Morphogenesis of Fetal Mouse Submandibular Glands by EGF

We have previously reported that epidermal growth factor (EGF) stimulates branching morphogenesis of the fetal mouse submandibular gland (SMG) (M. Kashimata and E. W. Gresik, 1997, Dev. Dyn. 208, 149–161) and that the EGF receptor (EGFR) is localized principally, if not exclusively, on the epithelial components of the fetal SMG (E. W. Gresik, M. Kashimata, Y. Kadoya, R. Mathews, N. Minami, and S. Yamashina, 1997, J. Histochem. Cytochem. 45, 1651–1657). The EGFR is a receptor tyrosine kinase, and after binding of its ligand, it triggers several intracellular signaling cascades, among them the one activating the mitogen-activated protein kinases (MAPK) ERK-1/2. Here we investigated whether EGF utilizes the ERK-1/2 signaling cascade to stimulate branching morphogenesis in the fetal mouse SMG. SMG rudiments were collected as matched pairs at E14, E16, and E18 (E0 = day of vaginal plug); placed into wells of defined medium (BGJb); and exposed to EGF for 5 or 30 min or to medium alone (controls). By Western blotting we found that EGF induced the appearance of multiple bands of phosphotyrosine-containing proteins, including bands at 170 kDa and 44 kDa/42 kDa, presumably corresponding to the phosphorylated forms of EGFR and ERK-1/2, respectively. Other blots showed the specific appearance of the phosphorylated EGFR and of phospho-ERK-1/2 in response to EGF. Immunohistochemical staining for phosphotyrosine increased at the plasma membrane after EGF stimulation for 5 or 30 min. Diffuse cytoplasmic staining for MEK-1/2 (the MAPK kinase that activates ERK-1/2) increased near the cell membrane after EGF stimulation. Phospho-ERK-1/2 was localized in the nuclei of a few epithelial cells after EGF for 5 min, but in the nuclei of many cells after EGF for 30 min. PD98059, an inhibitor of phosphorylation and activation of MEK-1/2, by itself inhibited branching morphogenesis and, furthermore, decreased the stimulatory effect of EGF on branching. Western blots confirmed that this inhibitor blocked phosphorylation of ERK-1/2 in fetal SMGs exposed to EGF. These results show that components of the ERK-1/2 signaling cascade are present in epithelial cells of the fetal SMG, that they are activated by EGF, and that inhibition of this cascade perturbs branching morphogenesis. However, EGF did not cause phosphorylation of two other MAPKs, SAPK/JNK or p38MAPK, in fetal SMGs. These results imply that the ERK-1/2 signaling is responsible, at least in part, for the stimulatory effect of EGF on branching morphogenesis of the fetal mouse SMG.     

miR-223促进小鼠胚胎成纤维细胞复制性衰老

微小RNA（MicroRNAs（或miRNAs）是作为强大的基因表达调控子,广泛参与多种生命过程,在细胞衰老进程中的作用也日益受到关注。miR-223是一个典型的抑癌基因,可显著抑制细胞增殖能力。此外,miR-223与阿尔茨海默症、心血管疾病以及类风湿性关节炎等衰老相关疾病的发生发展密切相关。尽管如此,miR-223在细胞衰老进程中的作用及其分子机制尚未见报道。本研究通过连续传代建立了小鼠胚胎成纤维细胞（MEF细胞）的复制性衰老模型,并利用荧光定量qRT-PCR检测发现,miR-223在衰老MEF细胞中的表达水平显著上调。随后,通过转染miR-223模拟物Agomir-223在MEF细胞中过表达miR-223,结果显示过表达miR-223可显著促进MEF细胞的衰老表型并抑制其增殖能力,而抑制miR-223的表达可延缓MEF细胞的复制性衰老进程。进一步利用生物信息学方法预测获得多个miR-223的候选衰老相关靶基因,包括Rasa1、Ddit4和Smad1等。然而双萤光素酶报告系统结果显示,miR-223并不显著影响其萤光强度,表明它们很可能并不是miR-223的下游靶基因。综上所述,miR-223可显著促进MEF细胞复制性衰老,然而其调节细胞衰老进程的分子机制依然有待深入研究。