三维重构展示类胚体样结构

背景：干细胞因其具有增殖能力及分化潜能,被认为是实施细胞治疗的重要细胞而被广泛研究。实现干细胞与组织三维结构的展示与分析,对阐明干细胞增殖与分化的机制有重要意义。 目的：探讨实现对经分化的类胚体样组织结构进行三维重构展示的方法。 方法：以来源于骨骼肌干细胞经诱导具有三维立体结构的类胚体作为实验对象,利用多色免疫荧光染色结合Z轴连续图像采集和三维重构技术,对经分化的类胚体样组织结构进行三维重构展示。 结果与结论：利用实验方法可实现对类胚体样组织结构进行三维重构,经重构的三维模型可实现任意角度旋转,利用不同荧光通道iso-surface处理后,选择分别显示不同颜色功能或任意横断面展示功能,即可展示心脏特异性肌钙蛋白Ⅰ阳性组织结构与细胞核的空间位置关系,并清晰展示在经分化形成类心肌组织的结构中存在细胞核。提示利用该技术,可对经分化的类胚体样组织结构进行三维重构展示及测量。     

Cell senescence,apoptosis and DNA damage cooperate in the remodeling processes accounting for heart morphogenesis

During embryonic development, organ morphogenesis requires major tissue rearrangements that are tightly regulated at the genetic level. A large number of studies performed in recent decades assigned a central role to programmed cell death for such morphogenetic tissue rearrangements that often sculpt the shape of embryonic organs. However, accumulating evidence indicates that far from being the only factor responsible for sculpting organ morphology, programmed cell death is accompanied by other tissue remodeling events that ensure the outcome of morphogenesis. In this regard, cell senescence has been recently associated with morphogenetic degenerative embryonic processes as an early tissue remodeling event in development of the limbs, kidney and inner ear. Here, we have explored cell senescence by monitoring β‐galactosidase activity during embryonic heart development where programmed cell death is believed to exert an important morphogenetic function. We report the occurrence of extensive cell senescence foci during heart morphogenesis. These foci overlap spatially and temporally with the areas of programmed cell death that are associated with remodeling of the outflow tract to build the roots of the great arteries and with the septation of cardiac cavities. qPCR analysis allowed us to identify a gene expression profile characteristic of the so‐called senescence secretory associated phenotype in the remodeling outflow tract of the embryonic heart. In addition, we confirmed local upregulation of numerous tumor suppressor genes including p21, p53, p63, p73 and Btg2. Interestingly, the areas of cell senescence were also accompanied by intense lysosomal activation and non‐apoptotic DNA damage revealed by γH2AX immunolabeling. Considering the importance of sustained DNA damage as a triggering factor for cell senescence and apoptosis, we propose the coordinated contribution of DNA damage, senescence and apoptotic cell death to assure tissue remodeling in the developing vertebrate heart.     

Quantitative Three‐Dimensional Analysis of Embryonic Chick Morphogenesis Via Microcomputed Tomography

Embryonic development is a remarkably complex and rapidly evolving morphogenetic process. Although many of the early patterning events have been well described, understanding the anatomical changes at later stages where clinically relevant malformations are more likely to be survivable has been limited by the lack of quantitative 3D imaging tools. Microcomputed tomography (Micro‐CT) has emerged as a powerful tool for embryonic imaging, but a quantitative analysis of organ and tissue growth has not been conducted. In this study, we present a simple method for acquiring highly detailed, quantitative 3D datasets of embryonic chicks with Micro‐CT. Embryos between 4 and 12 days (HH23 and HH40) were labeled with osmium tetroxide (OT), which revealed highly detailed soft tissue anatomy when scanned at 25 μm resolution. We demonstrate tissue boundary and inter‐tissue contrast fidelity in virtual 2D sections are quantitatively and qualitatively similar to those of histological sections. We then establish mathematical relationships for the volumetric growth of heart, limb, eye, and brain during this period of development. We show that some organs exhibit constant exponential growth (eye and heart), whereas others contained multiple phases of growth (forebrain and limb). Furthermore, we show that cardiac myocardial volumetric growth differs in a time and chamber specific manner. These results demonstrate Micro‐CT is a powerful technique for quantitative imaging of embryonic growth. The data presented here establish baselines from which to compare the effects of genetic or experimental perturbations. Quantifying subtle differences in morphogenesis is increasingly important as research focuses on localized and conditional effects. Anat Rec,, 2010. © 2010 Wiley‐Liss, Inc.     

Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity

Proper development requires coordination in growth of the cell types composing an organ. Many plant and animal cells are polyploid, but how these polyploid tissues contribute to organ growth is not well understood. We found the Drosophila melanogaster subperineurial glia (SPG) to be polyploid, and ploidy is coordinated with brain mass. Inhibition of SPG polyploidy caused rupture of the septate junctions necessary for the blood-brain barrier. Thus, the increased SPG cell size resulting from polyploidization is required to maintain the SPG envelope surrounding the growing brain. Polyploidization likely is a conserved strategy to coordinate tissue growth during organogenesis, with potential vertebrate examples.     

Quantitative volumetric analysis of cardiac morphogenesis assessed through micro-computed tomography.

We present a method to generate quantitative embryonic cardiovascular volumes at extremely high resolution without tissue shrinkage using micro-computed tomography (Micro-CT). A CT dense polymer (Microfil, Flow Tech, Inc.) was used to perfuse avian embryonic hearts from Hamburger and Hamilton stage (HH) 15 through HH36, which solidified to create a cast within the luminal space. Hearts were then scanned at 10.5 mum(3) voxel resolution using a VivaCT scanner, digital slices were contoured for regions of interest, and computational analysis was conducted to quantify morphogenetic parameters. The three-dimensional morphology was compared with that of scanning electron microscopy (SEM) images and serial section reconstruction of similarly staged hearts. We report that Microfil-perfused hearts swelled to maximum end-diastolic volume with negligible shrinking after polymerization. Comparison to SEM revealed good agreement of cardiac chamber proportions and intracardiac tissue structures (i.e., valves and septa) at the stages of development assessed. Quantification of changes in chamber volume over development revealed several notable results that confirm earlier hypotheses. Heart chamber volumes grow over two orders of magnitude during the 1-week developmental period analyzed. The atrioventricular canal comprised a significant proportion of the early heart volume. While left atrium/left ventricular volume ratios approached 1 in later development, right atrium/right ventricle ratios increase to over 2.5. Quantification of trabeculation patterns confirmed that the right and left ventricles are similarly trabeculated before HH27, after which the right ventricle became quantitatively coarser than that of the left ventricle. These results demonstrate that Micro-CT can be used to image and quantify cardiovascular structures during development.     

Cancer morphogenesis: role of mitochondrial failure

Adenosine triphosphate (ATP) required for normal cell metabolism is mainly supplied by mitochondrial oxidative phosphorylation (OXPHOS), which is limited by available oxygen and modulated by cell signaling pathways. Primary or secondary OXPHOS failure shifts cell metabolism towards ATP generation by glycolysis (Warburg effect). The objective of this paper is to clarify the role of mitochondrial dysfunction in cancer morphogenesis and to elucidate how faulty morphogen gradient signaling and inflammatory mediators that regulate OXPHOS can cause cancer-induced morphogenesis. Developmental morphogenesis and cancer morphogenesis are regulated by morphogenetic fields. The importance of morphogenetic fields is illustrated by transplantation of metastatic melanoma cells into the chick-embryo; the tumor cells adapt morphologies that resemble normal cells and function normally in the host. A morphogen gradient is a simple form of morphogenetic field. Morphogens such as those of the transforming growth factor (TGF)-beta family inhibit and stimulate basic cell proliferation at high and low concentrations respectively. Along a signaling gradient of declining TGF-beta concentration, with increasing distance from the gradient source, cell proliferation is first gradually less inhibited, and then gradually stimulated, thus generating a concave curved structure. In 3D cell cultures, TGF-beta concentration determines the diameter of the tubules it induces. TGF-beta1 can modulate mitochondrial OXPHOS via adenine nucleotide translocase (ANT) or uncoupling protein (UCP) via COX-2 and prostaglandin (PG) E2. Thus, gradients of TGF-beta can regulate the radius of curvature of tissues by modulating mitochondrial ATP generation. Derailment of morphogen control of mitochondrial ATP synthesis can lead to abnormal spatial variation in ATP supply, abnormal spatial distribution of cell proliferation, and cancer morphogenesis. Involvement of COX-2 in morphogen signaling is a mechanism whereby inflammation can promote carcinogenesis. Restoration of OXPHOS can reverse cancer morphogenesis and restore normal tissue morphology. Avoiding exposure to environmental mitochondrial toxins and toxic food ingredients should reduce the risk of cancer.     

Infants thinner at birth exhibit smaller kidneys for their size in late gestation in a sample of fetuses with appropriate growth

Fetal ultrasound measurements were employed to investigate the relationship between weight and ponderal index at birth and kidney size during the second (23 weeks) and third (32 weeks) trimesters of pregnancy in a sample of 25 normally growing fetuses. Kidney volume and kidney volume / fetal weight ratio at 32 weeks are significantly and positively related to both weight and ponderal index at birth, controlling for sex, gestational age at birth, and day of ultrasound measurement. A second‐degree polynomial relationship approximates the predictability of kidney volume fetal weight ratio at 23 weeks to that at 32 weeks, demonstrating shifting growth rates in fetal organ and body growth relationships during midgestation. Sex and parental size are suggested as contributing to these patterns. Females have a surge in renal growth between 23 and 32 weeks to catch up to earlier growing males, and maternal weight significantly predicts incremental growth in kidney volume and the kidney volume / fetal weight ratio at 32 weeks of gestation. The observation that fetuses relatively thin at birth have relatively smaller kidneys for their size in late gestation suggests that the influence of maternal weight on birth outcome may act through organ growth. Am. J. Hum. Biol. 14:398–406, 2002. © 2002 Wiley‐Liss, Inc.     

Concise review: applying stem cell biology to vascular structures

The vasculature, an organ that penetrates every other organ, is ideally poised to be the site where pools of stem cells are placed, to be deployed and committed in response to feedback regulation, and to respond to demands for new vascular structures. These pools of multipotent cells are often under the regulation of various members of the transforming growth factor-β superfamily, including the bone morphogenetic proteins and their antagonists. Regulation of stem cell populations affects their recruitment, differentiation, spatial organization, and their coordination with host tissue. Loss and dysregulation of feedback control cause a variety of diseases that involve ectopic tissue formation, including atherosclerotic lesion formation and calcification, diabetic vasculopathies, and arteriovenous malformations.     

Dynamics of follicular growth and atresia of large follicles during the ovarian cycle of the guinea pig: Fate of the degenerating follicles,a quantitative study

Background: A quantitative integrated study of healthy ovarian follicles of different sizes and their mitotic activity and of clearly defined atretic stages of involuting large growing follicles at different stages of the guinea pig ovarian cycle is not available in the literature. We considered that such a study would reveal new aspects of ovarian tissue dynamics and provide new information in an organ with a continuous phenotypic transformation of its cellular components. Methods: Ovaries from guinea pigs were removed on days 1 (opening of the vagina), 3, 6, 9, 13, and 16 of the cycle, and the following were measured in serial sections: (1) total number of healthy follicles falling into categories based on the volume occupied by granulosa cells, (2) total number of atretic follicles falling into clearly defined morphological stages of the degenerative and involutionary process affecting medium to large follicles, and (3) proportion of metaphase-arrested granulosa cells, after colcemid injection, in healthy follicles of different size categories. Results: Dynamic patterns of follicular growth and degeneration were revealed that permitted the following main conclusions and observations: (1) small to middle-size follicles can reach the maximal category mass of granulosa mass within 6–7 days, and the number of granulosa cells can increase 6–7-fold during this interval, (2) the cohort that gives rise to 2–6 preovulatory follicles and to the large follicles that will undergo atresia during each cycle varied from 68 to 108 follicles, (3) cell death starts in the granulosa cell layers of large follicles even when neighbouring cells maintain a high mitotic activity and it spreads rapidly; dead granulosa cells are cleare by nucleolysis and cytolysis in the absence of blood leucocytes or neovascularization, (4) foci of atresia are observed also in a few preovulatory follicles, (5) antral cavities of follicles with dead granulosa cells in the process of being lysed shrink and are filled within 2–3 days with large fibroblast-like cells arising from phenotypic transformation of inner layers of theca interna, with no evidence of mitotic activity or angiogenesis; the outer layers of theca interna involute, and by progressive atrophy and a process of cell death, minute nodular structures arise with remnants of the ovum and zona pellucida, and (6) a transient wave of degeneration affects a proportion of small and middle-size follicles during the metestrous period. This process does not resemble the morphological phenomenology of follicular involution, which affects only large follicles. Conclusions: This study contributes to a fuller understanding of the dynamics and time relationships of follicular growth and loss in the guinea pig ovary and provides new morphogenetic information on the atretic process. It would be valuable for the design of experiments on endocrine and paracrine interactions involved in follicular growth and atresia. © 1995 Wiley-Liss, Inc.     

The Role of Estradiol,Progesterone, and Transforming Growth Factor on Human Endometrioma Cell Culture

The present study demonstrates several aspects of endometrioma cells in culture, namely, 1) cell growth, proliferation, and morphology, 2) effect of cell culture on estrogen and progesterone receptor concentration, 3) effect of estradiol, progesterone, and transforming growth factor. The tissue sample was obtained from ovarian endometriomas that were removed via laparotomy or laparoscopy. The tissue sample was digested with collagenase. After washing, the tissue was cultured in endothelial cell culture medium. Cell count was done by flow cytometry. Receptor study was done by immunohistochemistry. The results demonstrated the growth and proliferation of endometrioma cell in culture medium. Electron microscopy showed stroma-like cells. The cells lost their estrogen and progesterone receptors. Estradiol and progesterone added to these cultures did not affect the rate of growth and proliferation of the cells. Transforming growth factor significantly increased the rate of growth and proliferation of these cells.     

Morphology and cell classification of large neurons in the adult human dentate nucleus: A quantitative study

The dentate nucleus represents the most lateral of the four cerebellar nuclei that serve as a major relay centres for fibres coming from the cerebellar cortex. Although many relevant findings regarding to the three-dimensional structure, the neuronal morphology and the cytoarchitectural development of the dentate nucleus have been presented so far, very little quantitative information has been collected to further explain several types of large neurons in the dentate nucleus. In this study we quantified the morphology of the large dentate neurons in the adult human taking, into account seven morphometric parameters that describe the main properties of the cell soma, the dendritic field and the dendritic branching pattern. Since the lateral cerebellar nucleus in the cat and other lower mammals is homologous to the dentate nucleus in primates and man, we have classified our sample of large neurons in accordance with the shape of the cell body, the dendritic arborisation and their location within the dentate nucleus. By performing the appropriate statistical analysis, we have proved that our sample of human dentate neurons can be classified into four distinct types. In that sense, our quantitative analysis verifies the validity of previous qualitative conclusions concerning the large neurons in the developing human dentate nucleus. Furthermore, the present study represents the first attempt to perform a quantitative analysis and cell classification of the large projection neurons in the adult human dentate nucleus.     

Replicative potential and the duration of the cell cycle in human fibroblasts: coordinate stimulation by epidermal growth factor.

The in vitro replicative potential of human diploid fibroblasts can be increased by polypeptide growth factors such as epidermal growth factor (EGF). Also, the cycle time of EGF-stimulated cells is, on average, decreased and their mitotic cell volume is reduced. Therefore, the regulation of cell size by the duration of the cell cycle may be one process which determines replicative potential. The growth response to a continuous presence of EGF, however, appears to be limited by eventual desensitization to the growth factor.     

不同组织部位诱导膜血管化及成骨因子表达的比较

文题释义 诱导膜技术：又称膜诱导技术、Masquelet技术,1986年由Masquelet等最先提出并使用,是一种重建超临界尺寸骨缺损的有效方法。该技术在清创后所形成的骨缺损处植入骨水泥占位器,以形成一层生物膜,并在所形成的膜内进行植骨对骨缺损进行二次重建。该技术修复大段骨缺损具有重建时间短、骨愈合率高、并发症发生率较低等优势,其疗效已经被广泛证实。 血管内皮生长因子：是一种生长因子,在血管生成中发挥重要作用。血管内皮生长因子与血管内皮表面的特异性受体结合,可以促进内皮细胞的增殖、迁移,并可增加局部毛细血管的通透性,促使纤维蛋白原渗出,为血管内皮细胞的迁移和血管形成提供基质。在促进骨愈合方面,血管内皮生长因子通过促进血管增生,协调软骨细胞的消长、细胞外基质的改建,加快软骨内成骨。血管内皮生长因子还可以趋化并促进成骨细胞的分化,使碱性磷酸酶活性增强,局部钙盐沉积增加,促进骨愈合。 背景：研究发现在皮下、肌肉等部位植入聚甲基丙烯酸甲酯骨水泥也可形成诱导膜,膜内同样具有微血管的形成并分泌多种成骨因子。 目的：比较皮下、肌肉、股骨骨缺损处诱导膜内血管化程度和成骨因子表达的差异。 方法：将36只雄性SD大鼠(购自广州中医药大学实验动物中心)随机分为3组,分别在后肢皮下、肌肉内、股骨骨缺损处植入聚甲基丙烯酸甲酯抗生素骨水泥占位器,每组12只。植入6周后,取出骨水泥周围的诱导膜,苏木精-伊红染色观察诱导膜组织形态结构变化,Western Blot印迹方法、RT-qPCR法、免疫组织化学染色法检测诱导膜中骨形态发生蛋白2、转化生长因子β1、血管内皮生长因子的表达情况。实验获得广州中医药大学动物实验伦理委员会批准,批准号：20181101006。 结果与结论：①苏木精-伊红染色显示3组均可形成诱导膜,但骨缺损组诱导膜组织切片外层血管数量较肌肉组、皮下组多,靠近骨水泥侧的内层成纤维细胞和肌纤维细胞数量较肌肉组、皮下组多;②免疫组织化学染色显示,骨缺损组骨形态发生蛋白2、转化生长因子β1、血管内皮生长因子阳性表达最多,皮下组最少;③Western Blot与RT-qPCR检测显示,骨缺损组骨形态发生蛋白2、转化生长因子β1、血管内皮生长因子表达均多于肌肉组、皮下组(P < 0.001);④结果表明,不同的周围组织条件对诱导膜的组织结构和成骨因子表达有重要影响,在骨缺损处植入聚甲基丙烯酸甲酯骨水泥可提高诱导膜的形成质量,膜内新生血管更丰富,骨生长因子的表达量更多。ORCID: 0000-0003-1405-0765(李树源) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Relationship between pituitary and adipose tissue after hypothalamic denervation in the female rat. A morphometric immunohistochemical study

Neonatal administration of monosodium glutamate (MSG) to rats produces severe lesions in certain hypothalamic nuclei, with repercussions in different neuroendocrine axes, and serves as a model for their study. In addition, adipose tissue, as a target organ, is known to be directly related to several neurondocrine axes. We used 21-day-old female Sprague-Dawley rats that had received a neonatal treatment with MSG (4 mg/g body weight, i.p., from day 2 up to day 10 of age) in addition to control rats (injected with 10% NaCl solution, on a similar schedule). We performed a specific immunohistochemical study on each anterior-pituitary cell population, along with the morphometry of these cells and of the parietal and visceral adipose tissue, and measured the levels of serum leptin and triglycerides. The MSG animals evinced significant changes in volume density (VD), cell density (CD), and cell size (CS) in the corticotropes, thyrotropes, and LH gonadotropes, but not in the somatotropes, lactotropes, and FSH gonadotropes. The modification common to the three cell types was a hyperplasia, but with different results depending on cell size. Furthermore, in the MSG rats significant changes were also observed in the VD, CD, and CS of the adipose tissue, consisting of adipogenesis and decrease of adipocyte size in visceral fat, together with probable lipogenesis as judged by an increase in adipocyte size in the parietal fat. The serum levels of leptin and triglycerides appeared significantly higher in MSG animals. For the first time in this animal model, and at the level of three neuroendocrine axes, our results suggest changes that correlate hypothalamic damage, cellular pituitary alterations, and the response of the adipose tissue as a target organ for MSG insult.     

Meningiomas: histogenesis and classification. A comparative morphological study

80 meningiomas were analyzed with various morphological methods including smear preparations, frozen section conventional histology. In all, the establishment of short term in-vitro growth was attempted and led to rapid cell proliferation in all but few exceptions. Electron microscopy was equally performed in the cases. In 37 of these meningiomas, commercially available antibodies against a whole pattern of tissue markers including cytokeratin, fibronectin, vimentin, S-100 protein and others were applied and visualized with the PAP method. The same was done with 4 in-vitro grown meningiomas. The sample of tumors comprised the major subtypes; age and sex distribution were consistent with known data. Electron microscopy showed only quantitative differences between different types, exhibiting as main features folded membranes with desmosomes and intermediate filaments. Cell types occurring in-vitro were dependent on the stage of proliferation. The tissue marker distribution showed vimentin as common to almost all meningiomas and fibronectin to half of them. Both antigens were observed after short term in-vitro growth in the tumor cells. It is concluded, that the central group of meningiomas despite of many different particular features has a common and uniform cellular make up demonstrated in all methods reported. The bearing of the intermediate mesodermal(neuro) ectodermal position of those tumors for their proper classification at the time being can only be discussed.     

Morphological heterogeneity of Paracoccidioides brasiliensis: relevance of the Rho-like GTPase PbCDC42

Paracoccidioides brasiliensis budding pattern and polymorphic growth were previously shown to be closely linked to the expression of PbCDC42 and to influence the pathogenesis of the fungus. In this work we conducted a detailed morphogenetic evaluation of the yeast-forms of 11 different clinical and environmental P. brasiliensis isolates comprising four phylogenetic lineages (S1, PS2, PS3 and Pb01-like), as well as a PbCDC42 knock-down strain. High variations in the shape and size of mother and bud cells of each isolate were observed but we did not find a characteristic morphologic profile for any of the phylogenetic groups. In all isolates studied, the bud size and shape were demonstrated to be highly dependent on the mother cell. Importantly, we found strong correlations between PbCDC42 expression and both the shape of mother and bud cells and the size of the buds in all isolates and the knock-down strain. Our results suggested that PbCDC42 expression can explain approximately 80% of mother and bud cell shape and 19% of bud cell size. This data support PbCDC42 expression level as being a relevant predictor of P. brasiliensis morphology. Altogether, these findings quantitatively describe the polymorphic nature of the P. brasiliensis yeast form and provide additional support for the key role of PbCDC42 expression on yeast cell morphology.     

In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method

Polycaprolactone (PCL) cylindrical scaffolds with gradually increasing pore size along the longitudinal direction were fabricated by a novel centrifugation method to investigate pore size effect on cell and tissue interactions. The scaffold was fabricated by the centrifugation of a cylindrical mold containing fibril-like PCL and the following fibril bonding by heat treatment. The scaffold showed gradually increasing pore size (from approximately 88 to approximately 405 microm) and porosity (from approximately 80% to approximately 94%) along the cylindrical axis by applying the centrifugal speed, 3000 rpm. The scaffold sections were examined for their in vitro cell interactions using different kinds of cells (chondrocytes, osteoblasts, and fibroblasts) and in vivo tissue interactions using a rabbit model (skull bone defects) in terms of scaffold pore sizes. It was observed that different kinds of cells and bone tissue were shown to have different pore size ranges in the scaffold for effective cell growth and tissue regeneration. The scaffold section with 380-405 microm pore size showed better cell growth for chondrocytes and osteoblasts, while the scaffold section with 186-200 microm pore size was better for fibroblasts growth. Also the scaffold section with 290-310 microm pore size showed faster new bone formation than those of other pore sizes. The pore size gradient scaffolds fabricated by the centrifugation method can be a good tool for the systematic studies of the interactions between cells or tissues and scaffolds with different pore size.