冬眠哺乳动物心血管机能特点

冬眠动物心血管系统适应于冬眠和激醒过程的特殊生理条件形成了其显著的机能稳定性。我们在总结已有研究的基础提出了冬眠动物心血管系统具有耐低体温,抗主律失常,耐缺氧三大机能特点。这些特点不仅是冬眠动物五非冬眠动物的区别,也是冬眠动物在冬眠季机能强化的方面。     

筛选转基因动物的新方法——PCR及其产物测序法

用特异性引物对肌球蛋白轻链2启动子(myosin light chain-2,MLC₂)-糜酶融合基因的转基因新生鼠鼠尾DNA进行PCR筛选, 低熔点琼脂糖凝胶电泳回收阳性样品PCR所扩出的DNA条带,纯化后用同一对引物中的一个进行单引物PCR测序,与所转外源基因序列比较,进一步确定整合有外源基因的阳性鼠.PCR及PCR 产物测序法检测转基因动物具有操作方便,灵敏度高及特异性强等优点.     

A Schwann cell matrix component of neuromuscular junctions and peripheral nerves

Molecules localized to the synapse are potential contributors to processes unique to this specialized region, such as synapse formation and maintenance and synaptic transmission. We used an immunohistochemical strategy to uncover such molecules by generating antibodies that selectively stain synaptic regions and then using the antibodies to analyse their antigens. In this study, we utilized a monoclonal antibody, mAb 6D7, to identify and characterize an antigen concentrated at frog neuromuscular junctions and in peripheral nerves. In adult muscle, immunoelectron microscopy indicates that the antigen is located in the extracellular matrix around perisynaptic Schwann cells at the neuromuscular junction and in association with myelinated and nonmyelinated axons in peripheral nerves. The maintenance of the mAb 6D7 epitope is innervation-dependent but is muscle-independent; it disappears from the synaptic region within 2 weeks after denervation, but persists after muscle damage when the nerve is left intact. mAb 6D7 immunolabelling is also detected at the neuromuscular junction in developing tadpoles. Biochemical analyses of nerve extracts indicate that mAb 6D7 recognizes a glycoprotein of 127 kDa with both N- and O-linked carbohydrate moieties. Taken together, the results suggest that the antigen recognized by mAb 6D7 may be a novel component of the synaptic extracellular matrix overlying the terminal Schwann cell. The innervation-sensitivity of the epitope at the neuromuscular junction suggests a function in the interactions between nerves and Schwann cells.     

Ultraviolet radiation,thiol reagents,and solubilization enhance AMPA receptor binding affinity via a common mechanism

The binding properties of membrane-bound or solubilized AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-type glutamate receptors from rat brain were tested following exposure to ultraviolet (UV)_radiation or incubation with the thiol reagent p-chloromercuriphenyl-sulfonic acid (PCMBS). Brief exposure to UV radiation (254 nm) increased [³H]AMPA binding to brain membranes, while binding to soluble fractions decreased. The increase in brain membrane binding was caused by an apparent interconversion of low-affinity [³H]AMPA binding sites into a higher-affinity state. Incubation with PCMBS caused a significant increase in [³H]AMPA binding to brain membranes but had no significant effect on [³HAMPA binding to solubilized receptors. There was an interaction between the PCMBS and UV effects in the brain membranes such that prior exposure to one of the treatments reduced the relative magnitude of the other's effects. The present results suggest that ultraviolet radiation, PCMBS and solubilization all increase AMPA receptor binding affinity via a common mechanism.     

肝星状细胞中表皮形态发生素表达调控机制及其作用研究

肝星状细胞是肝脏中重要的间质细胞,是肝细胞外基质的主要来源.表皮形态发生素(epimorphin、EPM、syntaxin2)在肝脏发育、再生及癌变过程中发挥了重要的作用,目前其表达变化的调控机制及对肝星状细胞的作用还未有报道.通过对肝组织标本进行检测,发现肝纤维化过程中肝星状细胞表达EPM上调.从表观遗传学的角度对EPM表达变化调控机制进行研究,发现DNA去甲基化促进了EPM的表达.为了研究EPM对肝星状细胞的可能的调节作用,将EPM表达质粒转染肝星状细胞,之后检测了EPM对肝星状细胞增殖及迁移能力的变化.结果证明EPM能够促进肝星状细胞的增殖与迁移.本研究发现,激活的肝星状细胞高表达EPM可能是由于DNA去甲基化引起的,同时,高表达的EPM能够促进肝星状细胞的增殖与迁移,进而促进肝纤维化进展.     

Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications

Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. Clinical applications of UC–MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2. UC–MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and quality control for their use in clinical applications. This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors. Blood vasculature was removed from UC. UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1–2 mm²) of UC membrane and Wharton’s jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic–antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in 5 % CO₂. The MSC properties of UC–MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC–MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice. Control cells consisted of UC–MSCs cultured in DMEM/F12 plus 1 % antibiotic–antimycotic, and 10 % fetal bovine serum (FBS). All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC–MSCs in these four groups exhibited similar marker profiles. Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes. The analysis of pluripotent cell markers showed UC–MSCs maintained the expression of the oncogenes Nanog and Oct4 after long term culture but failed to transfer tumors in NOD/SCID mice. Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC–MSCs that satisfy the minimum standards for clinical applications.     

Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch

TRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of “forefront” signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance.     

Interaction parameters between carbon nanotubes and water in Dissipative Particle Dynamics

Flow of water past an array of single-walled carbon nanotubes (SWNTs) is simulated in this work to determine the interaction parameters of carbon nanotubes (CNTs) and water using Dissipative Particle Dynamics (DPD). For this flow configuration, results from molecular dynamics simulations by Walther et al. are available and can be used for validation (Phys. Rev. E, 2004, 062201). The hydrodynamic properties for SWNT (32, 0) with diameter of 2.5 nm were determined in different Reynolds number flows. A set of appropriate DPD parameters was found so that the drag coefficients of the CNT agreed well with the Stokes–Oseen analytical solution and the fluid slip length on the CNT wall was comparable with the Walther et al. results. It was also found that it is feasible to apply these parameters in longer length and time scales by increasing the number of water molecules grouped into each DPD bead and still maintain the hydrodynamic properties of CNTs as well as their hydrophobic surface character.