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论文

    • 2015-10-06

    Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    요약 :
    Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function.
    Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serumstarved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-a, granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colonystimulating factor, and transforming growth factor (TGF)-b in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFNa, G-CSF, and TGF-b. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function

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    • 2015-10-06

    Quantification of early adipose-derived stem cell survival: comparison between sodium iodide symporter and enhanced green fluorescence protein imaging.

    요약 : 
    Objective: Strategies to overcome the problem of extensive early stem cell loss following transplantation requires a method to quantitatively assess their efficacy. This study compared the ability of sodium/iodide symporter (NIS) and enhanced green fluorescent protein (EGFP) imaging to monitor the effectiveness of treatments to enhance early stem cell survival.
    Methods: Human adipose-derived stem cells (ADSCs) transduced with an adenoviral vector to express both NIS and EGFP were mixed with culture media (control), matrigel (matrigel group) or pro-survival cocktail
    (PSC group), and 5×106 cells were injected into thigh muscles of C57BL/6 mice. Animals underwent serial optical imaging and 99mTcO4 – scintigraphy. Image-based EGFP fluorescence and 99mTcO4 – uptake was measured by region-of-interest analysis, and extracted tissues were measured for 99mTc activity. Fluorescent intensity measured from homogenized muscle tissue was used as reference for actual amount of viable ADSCs.
    Results: ADSCs were efficiently transduced to express EGFP and NIS without affecting proliferative capacity. The absence of significant apoptosis was confirmed by annexin V FACS analysis and Western blots for activated caspase-3. Both fluorescence optical imaging and 99mTcO4 – scintigraphy visualized implanted cells in living mice for up to 5days. However, optical imaging displayed large variations in fluorescence intensity, and thus failed to detect difference in cell survival between groups or its change over time. In comparison, 99mTcO4 -scintigraphy provided more reliable assessment of within-in group donor cell content as well as its temporal change. As a result, NIS imaging was able to discern beneficial effects of matrigel and pro-survival cocktail treatment on early ADSC survival, and provided quantitative measurements that correlated to actual donor cell content within implanted tissue.
    Conclusion: NIS reporter imaging may be useful for noninvasively assessing the efficacies of strategies designed to improve early survival of transplanted stem cell

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    • 2015-10-06

    Generation of red fluorescent protein transgenic dogs.

    요약 :
    Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in
    obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene-expressing
    construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFPfibroblasts. Using this approach, we produced the first
    generation of transgenic dogs with four female and two male expressing RFP

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    • 2015-10-06

    Recloned dogs derived from adipose stem cells of a transgenic cloned beagle

    요약 : 
    A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning

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    • 2015-10-06

    Generation of transgenic dogs that conditionally express green fluorescent protein

    요약 : 
    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP
    vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFPpositive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model
    will be useful for a variety of medical research studies

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    • 2015-10-06

    Lessons learned from cloning dogs.

    요약 : 
    The aim of this article is to review dog cloning research and to suggest its applications based on a discussion about the normality of cloned dogs. Somatic cell nuclear transfer was successfully used for production of viable cloned puppies despite limited understanding of in vitro dog embryo production. Cloned dogs have similar growth characteristics to those born from natural fertilization, with no evidence of serious adverse effects. The offspring of cloned dogs also have similar growth performance and health to those of naturally bred puppies. Therefore, cloning in domestic dogs can be applied as an assisted reproductive technique to conserve endangered species, to treat sterile canids or aged dogs, to improve reproductive performance of valuable individuals and to generate disease model animals

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