人骨髓基质细胞向成骨细胞分化过程中差异表达基因的筛选

为了探讨人骨髓基质细胞（bone marrow stromal cells,BMSCs）向成骨细胞分化过程中差异表达的基因,本实验采用体外培养人BMSCs,诱导向成骨细胞分化。分别选取培养12和21d的细胞作为驱动方（driver）和实验方（tester）,进行抑制消减杂交,构建cDNA消减文库,将挑选出的阳性克隆与GenBank人基因库中己公布的核酸序列进行同源性比较分析。结果表明,从培养21d的BMSCs中,筛查出5个差异基因,与人基因库中己知基因的同源性分别达到90％以上。有兴趣的是,核心蛋白聚糖和Bax inhibitorl在培养2ld的BMSCs中差异表达。RT-PCR检测显示,核心蛋白聚糖基因在培养21d的细胞中高表达,而在12d的细胞中未检测到表达;Bax inhibitorl基因在培养21d细胞中的表达明显高于12d的细胞。     

Targeted activation of androgen receptor signaling in the periosteum improves bone fracture repair

Low testosterone level is an independent predictor of osteoporotic fracture in elderly men as well as increased fracture risk in men undergoing androgen deprivation. Androgens and androgen receptor (AR) actions are essential for bone development and homeostasis but their linkage to fracture repair remains unclear. Here we found that AR is highly expressed in the periosteum cells and is co-localized with a mesenchymal progenitor cell marker, paired-related homeobox protein 1 (Prrx1), during bone fracture repair. Mice lacking the AR gene in the periosteum expressing Prrx1-cre (AR^-/Y;Prrx1::Cre) but not in the chondrocytes (AR^-/Y;Col-2::Cre) exhibits reduced callus size and new bone volume. Gene expression data analysis revealed that the expression of several collagens, integrins and cell adhesion molecules were downregulated in periosteum-derived progenitor cells (PDCs) from AR^-/Y;Prrx1::Cre mice. Mechanistically, androgens-AR signaling activates the AR/ARA55/FAK complex and induces the collagen-integrin α2β1 gene expression that is required for promoting the AR-mediated PDCs migration. Using mouse cortical-defect and femoral graft transplantation models, we proved that elimination of AR in periosteum of host mice impairs fracture healing, regardless of AR existence of transplanted donor graft. While testosterone implanted scaffolds failed to complete callus bridging across the fracture gap in AR^-/Y;Prrx1::Cre mice, cell-based transplantation using DPCs re-expressing AR could lead to rescue bone repair. In conclusion, targeting androgen/AR axis in the periosteum may provide a novel therapy approach to improve fracture healing.Subject terms: Bone development, Metabolic bone disease     

The role of Hedgehog signaling in fibrogenic liver repair

Repair of adult liver, like many tissues, involves the coordinated response of a number of different cell types. In adult livers, fibroblastic cells, ductular cells, inflammatory cells, and progenitor cells contribute to this process. Our studies demonstrate that the fates of such cells are dictated, at least in part, by Hedgehog, a fetal morphogenic pathway that was once thought to be active mainly during embryogenesis. Studies of injured adult human and rodent livers demonstrate that injury-related activation of the Hedgehog pathway modulates several important aspects of repair, including the growth of hepatic progenitor populations, hepatic accumulation of myofibroblasts, repair-related inflammatory responses, vascular remodeling, liver fibrosis and hepatocarcinogenesis. These findings identify the Hedgehog pathway as a potentially important target for biomarker development and therapeutic manipulation, and emphasize the need for further research to advance knowledge about how this pathway is regulated by and interacts with other signals that regulate adult liver repair.     

The adventures of Sonic Hedgehog in development and repair. I. Hedgehog signaling in gastrointestinal development and disease

Hedgehog (Hh) proteins are members of a family of secreted signaling factors that orchestrate the development of many organs and tissues including those of the gastrointestinal (GI) tract. The requirement for Hh activity is not limited to early development but underlies the homeostasis of a number of tissues, and abnormal activity of the Hh pathway is associated with several GI malignancies. Understanding the roles and mechanisms of action of Hh signaling both in development and postnatally should thus give novel insights into potential treatments for these diseases. Here we focus on the Hh signaling pathway and its role in GI tract development and maintenance and consider the diseases resulting from aberrant Hh activity.     

骨髓单个核细胞在周围神经修复中的应用

基础研究证实,多种细胞移植可以促进周围神经修复,其中来源丰富的骨髓单个核细胞,因具有取材过程简单、无交叉感染风险、无免疫排斥、可以自体移植等诸多优点,是目前重要的候选细胞之一。本文就近期有关骨髓单个核细胞的神经修复作用机制的研究、细胞植入修复受损周围神经的文献、以及与各种生物材料复合应用构建的组织工程化神经等方面最新进展进行综述,以期促进该领域基础向临床应用的转化。     

Articular cartilage repair with autologous bone marrow mesenchymal cells

Articular cartilage defects that do not repair spontaneously induce osteoarthritic changes in joints over a long period of observation. In this study, we examined the usefulness of transplanting culture‐expanded bone marrow mesenchymal cells into osteochondral defects of joints with cartilage defects. First, we performed experiments on rabbits and up on obtaining good results proceeded to perform the experiments on humans. Macroscopic and histological repair with this method was good, and good clinical results were obtained although there was no significant difference with the control group. Recent reports have indicated that this procedure is comparable to autologous chondrocyte implantation, and concluded that it was a good procedure because it required one step less than that required by surgery, reduced costs for patients, and minimized donor site morbidity. Although some reports have previously shown that progenitor cells formed a tumor when implanted into immune‐deficient mice after long term in vitro culture, the safety of the cell transplantation was confirmed by our clinical experience. Thus, this procedure is useful, effective, and safe, but the repaired tissues were not always hyaline cartilage. To obtain better repair with this procedure, treatment approaches using some growth factors during in vitro culture or gene transfection are being explored. J. Cell. Physiol. 225: 291–295, 2010. © 2010 Wiley‐Liss, Inc.     

Cell-type-related segregation of surface galactosyl-containing components at an early developmental stage in hemopoietic bone marrow cells in the rabbit

The avidin-biotin complex was used for the selective ultrastructural labeling of terminal cell surface galactosyl residues. Rabbit bone marrow cells were treated with the enzyme galactose oxidase in the presence of biotin hydrazide. Subsequent treatment with ferritin-avidin conjugates enabled the electron microscopic visualization of terminal membrane-based galactose and/or N-acetylgalactosamine on these cells. All stages of erythroid development were characterized by high levels of exposed cell surface galactose, whereas all leukoid cells in the same preparations were virtually unlabeled by the above method. Modulations in the distribution of these surface determinants during differentiation and maturation of rabbit erythroid cells were found to concur in inverse fashion with respect to that of terminal sialic acids. Neuraminidase treatment, before the above labeling procedure, resulted in the exposure of additional galactosyl residues on the surface of all bone marrow cell types. The results indicate that a galactose-bearing glycoconjugate(s) may comprise an erythroid-specific membrane constituent of rabbit bone marrow cells. The high density of galactose on the surface of even the earliest erythroid precursors may eventually enable the identification and isolation of a stem cell, which already contains the erythroid-specific galactoconjugate(s). The results suggest that variations in the spectrum of cell surface carbohydrates may serve as recognition signals in the complex set of intercellular interactions which occur during the development and maturation of the erythrocyte. The occurrence of similar but species- specific variations in the complement of surface heterosaccharides during erythroid development of humans and other mammals supports this contention.     

Hedgehog signaling induces cardiomyogenesis in P19 cells

Sonic Hedgehog (Shh) is a critical signaling factor for a variety of developmental pathways during embryogenesis, including the specification of left-right asymmetry in the heart. Mice that lack Hedgehog signaling show a delay in the induction of cardiomyogenesis, as indicated by a delayed expression of Nkx2-5. To further examine a role for Shh in cardiomyogenesis, clonal populations of P19 cells that stably express Shh, termed P19(Shh) cells, were isolated. In monolayer P19(Shh) cultures the Shh pathway was functional as shown by the up-regulation of Ptc1 and Gli1 expression, but no cardiac muscle markers were activated. However, Shh expression induced cardiomyogenesis following cellular aggregation, resulting in the expression of factors expressed in cardiac muscle including GATA-4, MEF2C, and Nkx2-5. Furthermore, aggregated P19 cell lines expressing Gli2 or Meox1 also up-regulated the expression of cardiac muscle factors, leading to cardiomyogenesis. Meox1 up-regulated the expression of Gli1 and Gli2 and, thus, can modify the Shh signaling pathway. Finally, Shh, Gli2, and Meox1 all up-regulated BMP-4 expression, implying that activation of the Hedgehog pathway can regulate bone morphogenetic protein signals. Taken together, we propose a model in which Shh, functioning via Gli1/2, can specify mesodermal cells into the cardiac muscle lineage.     

Hedgehog signaling regulates expansion of pancreatic epithelial cells

Embryonic Hedgehog signaling is essential for proper tissue morphogenesis and organ formation along the developing gastrointestinal tract. Hedgehog ligands are expressed throughout the endodermal epithelium at early embryonic stages but excluded from the region that will form the pancreas. Ectopic activation of Hedgehog signaling at the onset of pancreas development has been shown to inhibit organ morphogenesis. In contrast, Hedgehog signaling components are found within pancreatic tissue during subsequent stages of development as well as in the mature organ, indicating that a certain level of pathway activation is required for normal organ development and function. Here, we ectopically activate the Hedgehog pathway midway through pancreas development via expression of either Sonic (Shh) or Indian Hedgehog (Ihh) under control of the human Pax4-promoter. Similar pancreatic defects are observed in both Pax4-Shh and Pax4-Ihh transgenic lines, suggesting that regulation of the overall level of Hedgehog activity is critical for proper pancreas development. We also show that Hedgehog signaling controls mesenchymal vs. epithelial tissue differentiation and that pathway activation impairs formation of epithelial progenitors. Thus, tight control of Hedgehog pathway activity throughout embryonic development ensures proper pancreas organogenesis.     

Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice

The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.     

Clonal deletion of self-reactive T cells at the early stage of T cell development in thymus of radiation bone marrow chimeras

Sequential appearance of T cell subpopulations occurs in the thymocytes of irradiated C3H/He mice (H-2k, Mls-1b2a, Thy-1.2) after transplantation with bone marrow cells of AKR/J mice (H-2k, Mls-1a2b, Thy-1.1) (AKR----C3H chimeras). The donor-derived thymocytes of AKR----C3H chimeras on day 14 after bone marrow transplantation (BMT) contained a large number of blastlike CD4+CD8+ cells which represent relatively immature thymocytes, whereas those on day 21 after BMT consisted of small sized CD4+,CD8+ cells which represent a great part in normal thymocytes. To define the developmental stage at which clonal deletion of self-reactive T cells occurs in adult thymus, we followed the fate of V beta 6- or V beta 11-bearing T cells in the donor-derived thymocytes at the early stage of AKR----C3H chimeras. Mature thymocytes expressing high intensity of V beta 6 or V beta 11, which are involved in recognition of Mls-1a or MHC I-E gene products, respectively, were deleted from the donor-derived thymocytes on day 21. Immature thymocytes expressing low intensity of V beta 6 in CD3low thymocyte fraction decreased in proportion, whereas those expressing low intensity of V beta 11 rather increased in proportion in the donor-derived thymocytes of AKR----C3H chimeras from day 14 to day 21 after BMT. These results suggest that the clonal deletion of V beta 6-positive cells occurs just at the stage of immature CD3lowCD4+CD8+ cells, whereas the clonal deletion of V beta 11-positive cells may begin at the transitional stage from CD3lowCD4+CD8+ cells to CD3high single positive cells. Timing of negative selection of thymocytes may vary in distinct T cells capable of recognizing different self-Ag.     

Stromal cells of the bone marrow in growing bone

By means of electron microscopy, cytochemistry and radioautography with 3H-thymidine, the bone marrow stromal cells have been studied in the zones of endochondral osteogenesis in the rabbit and rat femoral bones. In the stromal cells demonstrating a high alkaline phosphatase activity are distinguished: perivascular, reticular fibroblastic, osteogenic cells. Populations of the perivascular phosphatase-positive cells include poorly differentiated DNA-synthesizing forms, as well as cells with signs of differentiation into stromal fibroblasts. Cleft-like spaces in cytoplasm of the fibroblastic reticular cells are, probably, formed as a result of lymphocyte-like mononuclears passing through. Phagocyting stromal elements are presented by macrophages, having perivascular localization and including into composition of erythroblastic islets. Mononuclear macrophages are revealed also on the surface of osseous trabecules, where they participate in destruction of hemopoetic and osteogenic cells.     

Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells.

Ghrelin was identified in the stomach as an endogenous ligand specific for the growth hormone secretagogue receptor (GHS-R). GHS-R is found in various tissues, but its function is unknown. Here we show that GHS-R is found in hepatoma cells. Exposure of these cells to ghrelin caused up-regulation of several insulin-induced activities including tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), association of the adapter molecule growth factor receptor-bound protein 2 with IRS-1, mitogen-activated protein kinase activity, and cell proliferation. Unlike insulin, ghrelin inhibited Akt kinase activity as well as up-regulated gluconeogenesis. These findings raise the possibility that ghrelin modulates insulin activities in humans.     

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hematopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen I, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis.     

Purinergic signaling in bone marrow stem cell mobilization

Purinergic Signalling -