老年大鼠神经组织星形胶质细胞的分选培养与炎性特征分析*

目的: 建立优化的年轻与老年大鼠神经组织星形胶质细胞的分离纯化方法,比较年轻大鼠与老年大鼠星形胶质细胞的形态、功能差异,探讨老化后星形胶质细胞的功能改变及其在衰老过程中发挥的可能机制。方法: 采用50%-35%的percoll密度梯度离心法分选年轻(2月龄)和老年(20月龄)SD大鼠的大脑与脊髓星形胶质细胞;每组细胞设置3个复孔,培养72 h后,采用免疫荧光检测星形胶质细胞特异性标志物胶质纤维酸性蛋白(GFAP),观察不同年龄阶段星形胶质细胞的形态特征;qPCR检测衰老标志(p16、p21)的表达,β-半乳糖苷酶染色检测星形胶质细胞的衰老情况;qPCR检测促炎因子(IL-1β、TNF-α)与抗炎因子(IL-10)的表达水平。结果: 采用50%-35%的percoll梯度分选得到的星形胶质细胞的数量多、活性好、纯度高达95%以上,可用于后续实验。与年轻大鼠神经组织的星形胶质细胞相比,分选自老年大鼠神经组织的星形胶质细胞在细胞形态上偏向激活态,突起较少;星形胶质细胞β-半乳糖苷酶染色阳性率升高,p16、p21表达也明显增多(P<0.01);老年大鼠神经组织的星形胶质细胞的促炎因子(IL-1β、TNF-α)表达升高(P<0.05),抗炎因子(IL-10)表达有所降低(P<0.05)。结论: 50%-35%的percoll梯度可以作为大鼠神经组织星形胶质细胞的分选纯化、原代培养的方法;随着年龄的增加,星形胶质细胞发生细胞老化,表现出促炎症表型,促进神经系统的炎性衰老,可能是神经系统老化及神经退行性疾病的机制之一。     

综述:星形胶质细胞介导的β-淀粉样蛋白代谢与阿尔茨海默病早期的关系

星形胶质细胞是中枢神经系统中含量最丰富的细胞,研究表明,星形胶质细胞与阿尔茨海默病(Alzheimer's disease,AD)病程有关,尤其是对AD主要致病蛋白β-淀粉样蛋白(β-amyloid protein,Aβ)的产生、内化和降解过程起着重要的调节作用．本文讨论星形胶质细胞中Aβ的产生,星形胶质细胞对Aβ的内化、降解和清除的机制,并阐释星形胶质细胞在Aβ代谢中的作用与AD早期发病机制的关系．     

PDAPP转基因小鼠脑组织内反应性星形胶质细胞的活化程度明显升高

目的　研究PDAPP转基因小鼠脑组织内反应性星形胶质细胞的活化程度。方法　通过免疫组织化学染色方法检测小鼠脑组织内反应性星形胶质细胞表达胶质纤维酸性蛋白 (GFAP)的情况 ,比较PDAPP转基因小鼠和C5 7 BL非转基因小鼠脑组织反应性星形胶质细胞的活化程度。结果　PDAPP转基因小鼠脑组织内反应性星形胶质细胞表达GFAP的水平明显高于C5 7 BL非转基因小鼠。结论　PDAPP转基因小鼠脑组织内存在明显的神经炎症反应     

星形胶质细胞在阿尔茨海默病中的功能变化及分子机制

星形胶质细胞在脑内数量最多,分布最广,对神经元有营养支持的作用,并且能够调控神经元的活性。越来越多的证据表明星形胶质细胞激活参与阿尔茨海默病(Alzheimer's disease,AD)的发生和发展。在AD病理情况下,星形胶质细胞在多种因子如β淀粉样蛋白(beta-amyloid,Aβ)和促炎细胞因子的作用下被激活,激活的星形胶质细胞进一步释放一氧化氮(Nitric oxide,NO)和多种炎性因子增强炎症级联反应。功能失常的星形胶质细胞会促进Aβ的产生,减弱对Aβ的摄取和清除,导致Aβ聚集沉积形成老年斑。激活的星形胶质细胞释放的炎症因子还能显著增加神经元内tau蛋白的异常过度磷酸化,产生神经纤维缠结。本文对星形胶质细胞在AD中参与神经变性的功能变化和分子机制进行总结,为星形胶质细胞作为靶点预防及治疗AD提供一定的理论依据。     

中间纤维的分子生物学

从头到脚,我们的身体是由富含称为中间纤维(IF)的胞内纤维蛋白的细胞组成的。免疫学和生物化学的证据表明在各种组织中组成IF的蛋白有五种:上皮内30种左右、分子量40—70 kd的角蛋白复杂群;肌肉內单一的52kd的结蛋白(desmin);间充质起源的细胞内单一的53kd的波形蛋白(vimentin),星形胶质细胞内单一的50kd的胶质纤维酸性蛋白(GFAP)和神经细胞内神经纤维蛋白三联体:NF-L(约65     

阿尔采末病中的炎症反应

阿尔采末病 (Alzheimer病 )是常见的老年病之一 ,其发病机制尚不明确 ,但已有大量相关的实验结果和假说 ,其中脑内的免疫炎症反应是一个重要的方面。小胶质细胞和星形胶质细胞是参与脑内炎症反应的主要免疫细胞。小胶质细胞可吞噬淀粉样蛋白 ,激活补体系统 ,释放大量炎性介质 ,激活星形胶质细胞。星形胶质细胞活化后参与淀粉样蛋白和其他病理沉积物的吞噬 ,也释放某些炎性细胞因子 ,部分反馈性抑制小胶质细胞活性。此外 ,脑内的炎症反应还与环氧酶、磷脂酶及自由基等密切相关。目前包括抑制胆碱脂酶活性、抗氧化、抑制淀粉样蛋白生成及聚集等在内的治疗方案均不能达到理想的疗效 ,而流行病学调查和临床实验回顾表明部分非甾体抗炎药可以降低其发病危险性 ,因此开发疗效更好、特异性更强、副作用低的抗炎药物是较有前景的方向     

MiR-144通过靶向抑制GRK5表达促进脊髓星形胶质细胞活化

已知miR-144与细胞活化和增殖有关,然而其具体分子机制尚不明确。本研究发现miR-144通过靶向GRK5促进脊髓星形胶质细胞的活化。运用real-time PCR检测脊髓损伤和正常大鼠的脊髓组织及其脊髓星形胶质细胞中miR-144的表达,发现与正常的组织和细胞相比,miR-144在脊髓损伤组织和星形胶质细胞中的表达水平显著降低;Western印迹检测到脊髓损伤大鼠的星形胶质细胞中GFAP蛋白的表达显著低于正常大鼠,而GRK5蛋白的表达高于正常大鼠;MTT分析结果显示转染miR-144可显著提高脊髓损伤大鼠的星形胶质细胞活性,但对细胞增殖无明显作用;酶活性试剂盒分析发现miR-144显著提高了SOD和GSH活性;生物学信息分析和萤光素酶报告基因检测结果显示miR-144能靶向结合GRK5,并下调GRK5的表达;MiR-144 mimic转染或miR-144 mimic与pcDNA-GRK5共转染脊髓损伤的星形胶质细胞,发现miR-144转染能通过激活NF-κB通路消除pcDNA-GRK5引起的细胞活化抑制。综上所述,miR-144通过靶定结合癌基因GRK5来促进脊髓星形胶质细胞细胞的活化。     

MiR-144通过靶向抑制GRK5表达促进脊髓星形胶质细胞活化北大核心CSCD

尽管miR-144与细胞活化、增殖有关,但其对脊髓星形胶质细胞的作用尚不明确。本文旨在探究正常和脊髓损伤(spinal cord injury,SCI)组织和细胞中miR-144表达,以及miR-144能否通过靶向调节G蛋白偶联受体激酶5(GRK5)上调脊髓星形胶质细胞活化。实时定量PCR(RT-q PCR)和Western印迹结果揭示,与正常的组织/细胞相比,miR-144在脊髓损伤组织和星形胶质细胞中的表达水平显著降低,而GRK5的表达升高;脊髓损伤大鼠的星形胶质细胞中胶质原纤维酸性蛋白(GFAP)的表达显著低于正常大鼠,而GRK5蛋白的表达高于正常大鼠;MTT分析结果显示,转染miR-144可显著提高脊髓损伤大鼠的星形胶质细胞活性,但对细胞增殖无明显作用;酶活性分析发现,miR-144显著提高SOD和GSH活性;萤光素酶报告基因检测结果证明,miR-144能靶向结合GRK5,下调GRK5表达。miR-144 mimic转染或miR-144 mimic与pc DNA-GRK5共转染脊髓损伤的星形胶质细胞后,我们发现,miR-144转染可通过激活NF-κB通路消除GRK5对细胞活化的抑制作用。综上所述,miR-144通过靶向抑制GRK5促进脊髓星形胶质细胞的活化。     

MiR-144通过靶向抑制GRK5表达促进脊髓星形胶质细胞活化北大核心CSCD

尽管miR-144与细胞活化、增殖有关,但其对脊髓星形胶质细胞的作用尚不明确。本文旨在探究正常和脊髓损伤(spinal cord injury,SCI)组织和细胞中miR-144表达,以及miR-144能否通过靶向调节G蛋白偶联受体激酶5(GRK5)上调脊髓星形胶质细胞活化。实时定量PCR(RT-q PCR)和Western印迹结果揭示,与正常的组织/细胞相比,miR-144在脊髓损伤组织和星形胶质细胞中的表达水平显著降低,而GRK5的表达升高;脊髓损伤大鼠的星形胶质细胞中胶质原纤维酸性蛋白(GFAP)的表达显著低于正常大鼠,而GRK5蛋白的表达高于正常大鼠;MTT分析结果显示,转染miR-144可显著提高脊髓损伤大鼠的星形胶质细胞活性,但对细胞增殖无明显作用;酶活性分析发现,miR-144显著提高SOD和GSH活性;萤光素酶报告基因检测结果证明,miR-144能靶向结合GRK5,下调GRK5表达。miR-144 mimic转染或miR-144 mimic与pc DNA-GRK5共转染脊髓损伤的星形胶质细胞后,我们发现,miR-144转染可通过激活NF-κB通路消除GRK5对细胞活化的抑制作用。综上所述,miR-144通过靶向抑制GRK5促进脊髓星形胶质细胞的活化。     

凝血酶对大鼠星形胶质细胞组织因子活性表达的影响及其机制

目的：观察凝血酶对大鼠星形胶质细胞组织因子活性表达的影响及其机制。方法;培养Ｗｉｓｔａｒ乳鼠星形胶质细胞,细胞用抗神经胶质纤维酸性蛋白单克隆抗体确认。细胞冻融液组织因子活性检测采用一期血浆复地。结果：与对照组比较,凝血酶使星形胶质细胞组织因子表达明显增高（Ｐ〈０．０５）。当凝血酶与三氟吡啦嗪或Ｈ７联用时,它对呈形胶质细胞表达组织因子活性的刺激作用受到抑制。与凝血酶组比较,已呵可碱十凝血酶组星形胶质     

Developmental expression and cellular origin of the laminin alpha2, alpha4, and alpha5 chains in the intestine.

Laminins are extracellular matrix glycoproteins that are involved in various cellular functions, including adhesion, proliferation, and differentiation. In this study, we examine the expression patterns and the cellular origins of the laminin alpha2, alpha4, and alpha5 chains in the developing mouse intestine and in in vitro mouse/chick or chick/mouse interspecies hybrid intestines. In situ hybridization and Northern blot analysis revealed that mRNA levels for all three laminin alpha chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin alpha4 mRNA and polypeptide are associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin alpha2 and alpha5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin alpha chains occur in the smooth muscle basement membrane, with a differential expression of laminin alpha5 chain in the circular and longitudinal smooth muscle layers. The cellular origin of laminin alpha2 and alpha5 chains found in the subepithelial cell basement membrane was studied by immunocytochemical analysis of mouse/chick or chick/mouse interspecies hybrid intestines at various stages of development using mouse-specific antibodies. Laminin alpha2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, while the laminin alpha5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern. We conclude that (1) multiple laminin alpha chains are expressed in the intestine, implying specific roles for individual laminin isoforms during intestinal development, and (2) reciprocal epithelial/mesenchymal interactions are essential for the formation of a structured subepithelial basement membrane.     

Intracellular signaling cascades triggered by the NK1 fragment of hepatocyte growth factor in human prostate epithelial cell line PNT1A

Hepatocyte Growth Factor (HGF)/c-MET signaling has an emerging role in promoting cell proliferation, survival, migration, wound repair and branching in a variety of cell types. HGF plays a crucial role as a mediator of stromal–epithelial interactions in the normal prostate but the precise biological function of HGF/c-Met interaction in the normal prostate and in prostate cancer is not clear. HGF has two naturally occurring splice variants and NK1, the smallest of these HGF variants, consists of the HGF amino terminus through the first kringle domain. We evaluated the intracellular signaling cascades and the morphological changes triggered by NK1 in human prostate epithelial cell line PNT1A which shows molecular and biochemical properties close to the normal prostate epithelium. We demonstrated that these cells express a functional c-MET, and cell exposure to NK1 induces the phosphorylation of tyrosines 1313/1349/1356 residues of c-MET which provide docking sites for signaling molecules. We observed an increased phosphorylation of ERK1/2, Akt, c-Src, p125FAK, SMAD2/3, and STAT3, down-regulation of the expression of epithelial cell–cell adhesion marker E-cadherin, and enhanced expression levels of mesenchymal markers vimentin, fibronectin, vinculin, α-actinin, and α-smooth muscle actin. This results in cell proliferation, in the appearance of a mesenchymal phenotype, in morphological changes resembling cell scattering and in wound healing. Our findings highlight the function of NK1 in non-tumorigenic human prostatic epithelial cells and provide a picture of the signaling pathways triggered by NK1 in a unique cell line.     

Peculiarities of the extracellular matrix in the interstitium of the renal stem/progenitor cell niche

The development of the nephron is piloted by interactions between epithelial and surrounding mesenchymal stem/progenitor cells. Data show that an astonishingly wide interstitial space separates both kinds of stem/progenitor cells. A simple contrasting procedure was applied to visualize features that keep renal epithelial and mesenchymal stem/progenitor cells in distance. The kidney of neonatal rabbits was fixed in solutions containing glutaraldehyde (GA) in combination with alcian blue, lanthanum, ruthenium red, or tannic acid. To obtain a comparable view to the renal stem/progenitor cell niche, the tissue was exactly orientated along the axis of collecting ducts. Fixation with GA or in combination with alcian blue or lanthanum revealed an inconspicuous interstitial space. In contrast, fixation with GA containing ruthenium red exhibits strands of extracellular matrix lining from epithelial stem/progenitor cells through the interstitium up to the surface of mesenchymal stem/progenitor cells. Fixation with GA containing tannic acid shows that the basal lamina of epithelial stem/progenitor cells, the adjacent interstitial space and also the surface of mesenchymal stem/progenitor cells are connected over a net of extracellular matrix. The applied technique appears to be a suitable method to illuminate the interstitium in stem/progenitor cell niches of specialized tissues, the microenvironment of tumors and extension of degeneration.     

STUDIES IN THE DYNAMICS OF HISTOGENESIS : II. TENSION OF DIFFERENTIAL GROWTH AS A STIMULUS TO MYOGENESIS IN THE ESOPHAGUS.

Esophageal Development. 1. The region of most active mitosis per mm. of cross-section in the esophagus is the entodermal epithelial tube. The mitotic figures follow a spiral path in the manner of a left-handed helix from the cephalic to the caudal direction. 2. The region of least active growth per mm. of cross-section in the esophagus is the mesenchyme surrounding the epithelial tube. 3. The helicoidal activity of the epithelial tube causes a vortical reaction in the surrounding mesenchyme. The mesenchymal whirlpool represents a reaction to the spirally grooving epithelial tube. 4. In embryos 9.5 to 14 mm. in length the esophageal epithelial tube grows relatively more rapidly in width than in length. During this period the myoblasts which form the inner, close spiral, muscle coat of the esophagus are becoming rapidly differentiated in the outer condensed margin of the mesenchymal maelstrom. 5. The nuclei, first spherical then oval, and finally rod shaped with rounded ends, are drawn out in the direction of the circumference of the mesenchymal rim which is directed tangentially. 6. The cytoplasm is also drawn out in the direction of the mesenchymal rim of the vortex. The elongated rows of isolated granules appear which subsequently, by confluence, form the myofibrillæ. These cytoplasmic derivatives are elongated in the direction of the circumference of the vortex. 7. Between the epithelial tube and the myoblastic rim at the periphery of the mesenchymal whorl is found the embryonic connective tissue. From this direct observation the conclusion is made that an optimum tensional stress stimulus is necessary to elicit the formation of muscular tissue at the circumference of the mesenchymal vortex. Consequently, the formation of a specific derivative from a pluripotent mesenchymal cell is due to the fortuitous circumstance of position. 8. In embryos from 14 to 24 mm. in length, the esophagus grows relatively more rapidly in length than in width. This elongation is due to two factors; first, the descent of the stomach, and, second, the resistance to diametrical growth presented by the inner close spiral musculature. The epithelial tube, still the dominant zone of mitotic activity, pursues the lines of least resistance, and consequently growth in length takes place. This is due to the shifting of the planes of cell division on account of the compression of the inner, close spiral, muscle coat. 9. The undifferentiated mesenchyme peripherad to the inner, close spiral musculature is elongated and the histogenetic changes in muscular formation are gradually taking place between 14 and 24 mm. A very attenuated, outer, elongated, spiral, or longitudinal muscle coat is detected in the esophagus of a 24 mm. pig embryo. 10. The characteristic intestino-colic flexure is a torsional reaction of the mesenchyme. The mesenchymal cells are thrown into a left-handed helicoidal series, corresponding to the activity in the epithelial tube. The right-handed helicoidal reaction of the mesenchyme, therefore, is due to the left-handed helicoidal growth of the epithelial tube. 11. The normal asymmetry of the abdominal viscera as well as the position of the gut is dependent upon the clockwise reaction of the stretched mesenchymal cell. These cells are stretched by the left-handed helicoidal growth of the epithelial tube. One factor producing situs inversus viscerum could be the reversal of the spiral growth of the epithelial tube resulting in a reaction of the mesenchyme in a direction opposite, namely counterclockwise, to that which occurs normally.     

Method for Obtaining Committed Adult Mesenchymal Precursors from Skin and Lung Tissue

Aims

The present study reports an easy and efficient method for obtaining adult mesenchymal precursors from different adult mouse tissues.

Materials and Methods

We describe the isolation and expansion of mesenchymal precursors from skin and lung by a non-enzymatic method. Skin and lung mesenchymal precursors isolated by a modified explant technique were characterized in vitro by defined morphology and by a specific gene expression profile and surface markers.

Results and Conclusions

Our results show that these precursors express stem cell and mesenchymal surface markers as well as epithelial markers. However, they are negative for markers of endothelium, cardiac and skeletal muscle or adipose tissue, indicating that they have initiated commitment to the tissues from which were isolated. These precursors can migrate without any stimulus and in response to stimuli as SDF1, MCP1 and TNFα and can be differentiated into epithelial lineages. Based on the properties of these precursors from adult tissues, we propose their use as tools for regenerative biomedicine.     

A Pituitary Homeobox 2 (Pitx2):microRNA-200a-3p:β-catenin Pathway Converts Mesenchymal Cells to Amelogenin-expressing Dental Epithelial Cells

Pitx2, Wnt/β-catenin signaling, and microRNAs (miRs) play a critical role in the regulation of dental stem cells during embryonic development. In this report, we have identified a Pitx2:β-catenin regulatory pathway involved in epithelial cell differentiation and conversion of mesenchymal cells to amelogenin expressing epithelial cells via miR-200a. Pitx2 and β-catenin are expressed in the labial incisor cervical loop or epithelial stem cell niche, with decreased expression in the differentiating ameloblast cells of the mouse lower incisor. Bioinformatics analyses reveal that miR-200a-3p expression is activated in the pre-ameloblast cells to enhance epithelial cell differentiation. We demonstrate that Pitx2 activates miR-200a-3p expression and miR-200a-3p reciprocally represses Pitx2 and β-catenin expression. Pitx2 and β-catenin interact to synergistically activate gene expression during odontogenesis and miR-200a-3p attenuates their expression and directs differentiation. To understand how this mechanism controls cell differentiation and cell fate, oral epithelial and odontoblast mesenchymal cells were reprogrammed by a two-step induction method using Pitx2 and miR-200a-3p. Conversion to amelogenin expressing dental epithelial cells involved an up-regulation of the stem cell marker Sox2 and proliferation genes and decreased expression of mesenchymal markers. E-cadherin expression was increased as well as ameloblast specific factors. The combination of Pitx2, a regulator of dental stem cells and miR-200a converts mesenchymal cells to a fully differentiated dental epithelial cell type. This pathway and reprogramming can be used to reprogram mesenchymal or oral epithelial cells to dental epithelial (ameloblast) cells, which can be used in tissue repair and regeneration studies.