新生鼠胰岛干细胞分离、培养和分化的实验研究

目的　探索新生鼠胰岛干细胞体外分离鉴定、培养以及体外分化的方法。方法　用胶原酶消化新生大鼠胰腺 ,将消化的组织碎片在pH7.4～ 7.6条件下经含血清RPMI 164 0及无血清RPMI164 0 (添加bFGF ,EGF ,N 2 )培养液中培养 ,观察形成类胰岛样细胞团的全过程。用胰岛素释放实验检测胰岛功能 ,免疫荧光法检测nestin的表达。结果　胰腺消化碎片培养 3 6h内 ,可见nestin阳性细胞贴壁 ,添加bFGF ,EGF ,N2后nestin阳性细胞快速增长 ,18～ 2 4d形成类胰岛样细胞团 ,并可表达胰岛素。结论　胰腺细胞中nestin阳性细胞具有胰岛干细胞特点 ,经体外培养可获得类胰岛样细胞团     

Smoking Alters Inflammation and Skeletal Stem and Progenitor Cell Activity During Fracture Healing in Different Murine Strains

Smokers are at a higher risk of delayed union or nonunion after fracture repair. Few specific interventions are available for prevention because the molecular mechanisms that result in these negative sequelae are poorly understood. Murine models that mimic fracture healing in smokers are crucial in further understanding the local cellular and molecular alterations during fracture healing caused by smoking. We exposed three murine strains, C57BL/6J, 129X1/SvJ, and BALB/cJ, to cigarette smoke for 3 months before the induction of a midshaft transverse femoral osteotomy. We evaluated fracture healing 4 weeks after the osteotomy using radiography, micro-computed tomography (μCT), and biomechanical testing. Radiographic analysis demonstrated a significant decrease in the fracture healing capacity of smoking 129X1/SvJ mice. μCT results showed delayed remodeling of fracture calluses in all three strains after cigarette smoke exposure. Biomechanical testing indicated the most significant impairment in the functional properties of 129X1/SvJ in comparison with C57BL/6J and BALB/cJ mice after cigarette smoke exposure. Thus, the 129X1/SvJ strain is most suitable in simulating smoking-induced impaired fracture healing. Furthermore, in smoking 129X1/SvJ murine models, we investigated the molecular and cellular alterations in fracture healing caused by cigarette smoking using histology, flow cytometry, and multiplex cytokine/chemokine analysis. Histological analysis showed impaired chondrogenesis in cigarette smoking. In addition, the important reparative cell populations, including skeletal stem cells and their downstream progenitors, demonstrated decreased expansion after injury as a result of cigarette smoking. Moreover, significantly increased pro-inflammatory mediators and the recruitment of immune cells in fracture hematomas were demonstrated in smoking mice. Collectively, our findings demonstrate the significant cellular and molecular alterations during fracture healing impaired by smoking, including disrupted chondrogenesis, aberrant skeletal stem and progenitor cell activity, and a pronounced initial inflammatory response. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Bone From Blood: Characteristics and Clinical Implications of Circulating Osteogenic Progenitor (COP) Cells

Circulating osteogenic progenitor (COP) cells are a population of cells in the peripheral blood with the capacity for bone formation, as well as broader differentiation into mesoderm-like cells in vitro. Although some of their biological characteristics are documented in vitro, their role in diseases of the musculoskeletal system remains yet to be fully evaluated. In this review, we provide an overview of the role of COP cells in a number of physiological and pathological conditions, as well as identify areas for future research. In addition, we suggest possible areas for clinical utilization in the management of musculoskeletal diseases. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Intercellular Interactions of an Adipogenic CXCL12-Expressing Stromal Cell Subset in Murine Bone Marrow

Bone marrow houses a multifunctional stromal cell population expressing C-X-C motif chemokine ligand 12 (CXCL12), termed CXCL12-abundant reticular (CAR) cells, that regulates osteogenesis and adipogenesis. The quiescent pre-adipocyte-like subset of CXCL12⁺ stromal cells (“Adipo-CAR” cells) is localized to sinusoidal surfaces and particularly enriched for hematopoiesis-supporting cytokines. However, detailed characteristics of these CXCL12⁺ pre-adipocyte-like stromal cells and how they contribute to marrow adipogenesis remain largely unknown. Here we highlight CXCL12-dependent physical coupling with hematopoietic cells as a potential mechanism regulating the adipogenic potential of CXCL12⁺ stromal cells. Single-cell computational analyses of RNA velocity and cell signaling reveal that Adipo-CAR cells exuberantly communicate with hematopoietic cells through CXCL12-CXCR4 ligand-receptor interactions but do not interconvert with Osteo-CAR cells. Consistent with this computational prediction, a substantial fraction of Cxcl12-creER⁺ pre-adipocyte-like cells intertwines with hematopoietic cells in vivo and in single-cell preparation in a protease-sensitive manner. Deletion of CXCL12 in these cells using Col2a1-cre leads to a reduction of stromal-hematopoietic coupling and extensive marrow adipogenesis in adult bone marrow, which appears to involve direct conversion of CXCL12⁺ cells to lipid-laden marrow adipocytes without altering mesenchymal progenitor cell fates. Therefore, these findings suggest that CXCL12⁺ pre-adipocyte-like marrow stromal cells prevent their premature differentiation by maintaining physical coupling with hematopoietic cells in a CXCL12-dependent manner, highlighting a possible cell-non-autonomous mechanism that regulates marrow adipogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).     

HNE1细胞株光动力学治疗前后形态学的动态变化

目的 探讨体外光动力学治疗（ＰＤＴ）引起鼻咽癌（ＮＰＣ）细胞死亡的作用机制。方法对鼻咽癌细胞株ＨＮＥ１体外光动力学治疗前后形态学变化进行动态观察。结果 对照组ＨＮＥ１细胞培养３ｈ,６ｈ,１２ｈ,２４ｈ和４８ｈ后细胞形态与正常培养组ＨＮＥ１细包开始变形皱缩,细胞界限模糊,细胞透光度和立体感较差,治疗２４～４８ｈ后细胞发生凋亡。结论 体外光动力学治疗对ＨＮＥ１具有明显的杀伤效应并ＨＮＥ１细胞凋亡。ＰＤ     

A high-density PEG interfacial layer alters the response to an EGF tethered polydimethylsiloxane surface

Previously, epidermal growth factor (EGF)-modified surfaces have shown promise in supporting cellular growth and adhesion on synthetic polymeric substrates. Surfaces prepared using a novel modification technique were investigated in the current work for their ability to support corneal epithelialization, important to the integration of a synthetic artificial cornea. EGF could be tethered to PDMS surfaces via a high-density, hetero-bifunctional PEG-NSC linking layer with a tunable surface concentration of up to 300 ng/cm². Only a small fraction of the EGF on these surfaces could be removed with SDS rinsing, indicative of covalent tethering. Studies with human corneal epithelial cells suggest a relatively linear increase in the number of corneal epithelial cells with increasing EGF concentration at all times. However, confluence was not achieved at any time point. It is believed that the presence of the non-adsorbent PEG layer, useful for preventing non-specific adsorption of proteins, may limit the cellular response by minimizing the adsorption of adhesion molecules. The effects of the EGF alone are clearly not sufficient to result in epithelialization of an artificial cornea surface. Altering both the adhesion and growth of corneal epithelial cells in a controlled manner may be necessary for epithelialization of an artificial cornea.     

Modulating the Activities of Human Mesenchymal Stem Cells (hMSCs) and C3A/HepG2 Hepatoma Cells by Modifying the Surface Characteristics of Poly(3-hydroxybutyrate-co-3-hydroxyhexnoate) (PHBHHx)

The new biodegradable polyester poly(3-hydroxybutyrate-co-3-hydroxyhexnoate) (PHBHHx) has a potential application in tissue engineering. The aim of this study was to present a deeper picture of the relationship between the cellular behavior and the surface characteristics of PHBHHx films. The pristine PHBHHx film was prepared by adopting the compression-molding method, and then the acrylic acid molecules were grafted on PHBHHx membrane surface by UV irradiation. The hydrophilic nature and surface roughness of various PHBHHx films were controlled by adjusting the acrylic acid concentration and the UV irradiation time. Although the surface characteristics of various PHBHHx films could not affect the metabolic activity of hMSCs, the performance of morphology of hMSCs was deeply affected by the hydrophilic nature and the orientation of surface scars. The hydrophilic nature would effectively improve the spread of hMSCs, and the orientation of surface scars would guide the growth direction of cytoskeleton (actin) inside hMSCs. In contrast, the behaviors of C3A/HepG2 hepatoma cells presented an opposite outcomes. Those surface characteristics were obviously associated with the performance of metabolic activity of C3A cells, but not with the morphology of C3A cells. Both hMSCs and C3A cells have unique cellular characteristics; therefore, their responses to environmental stimulations are significantly different.     

Fabrication and Characterization of Hydroxyapatite-Coated Polystyrene Disks for Use in Osteoprogenitor Cell Culture

A simple method is reported for fabricating polystyrene disk inserts coated with biomimetic carbonated hydroxyapatite (cHA) to be used for culturing osteoprogenitor cells or other stem cells. Roughened disks cut from tissue-culture polystyrene (TCPS) were coated in simulated body fluid with 5 × normal physiologic ionic concentrations (SBFx5) by a 2-step, 2-day method. The coatings were rigorously characterized by various methods and assessed in cell culture. An adherent, nearly 10 mm thick, relatively uniform layer of single-phase cHA was formed in two days. MC3T3-E1 and mouse calvaria-derived osteoprogenitor cells (pCOBs) were cultured on the cHA for various time points. Despite less initial attachment of both cell types to the cHA, proliferation rates on cHA were similar to that on TCPS. Two-fold greater cell attachment (P < 0.05) of the MC3T3-E1 cells was observed relative to the pCOBs, on both the TCPS and the cHA. Importantly, the coatings were relatively smooth, without the extensive agglomerates observed in other studies and remained adherent and morphologically unchanged after 21 days of culture. This technique can be used to rapidly produce high-quality cHA-coated TCPS disks for cell-culture studies.     

Targeting of Mesenchymal Stromal Cells by Cre‐Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells

The targeting specificity of tissue‐specific Cre‐recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn‐Cre and Dmp1‐Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high‐resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12^gfp mice to identify Cxcl12‐abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn‐Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1‐Cre also targets a subset of CAR cells, in which expression of osteoblast‐lineage genes is enriched. Finally, we introduce a new tissue‐specific Cre‐recombinase, Tagln‐Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn‐Cre and Dmp1‐Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research.