Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background

The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells.

Methods

A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses.

Results

Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles.

Conclusion

We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.     

Reproductive performance and expression of imprinted genes in somatic cell cloned boars

To assess the performance of boars derived by somatic cell cloning, we analyzed various aspects of their reproductive characteristics and the expression of two imprinted genes. Cloned boars (cloned Duroc × Jinhua) were analyzed for birth weight, growth rate, age at first ejaculation, semen characteristics and fertility, in comparison with naturally bred control boars of the same strain. The expression of imprinted genes was analyzed using the microsatellite marker SWC9 for the paternally expressed gene insulin‐like growth factor ‐2 (IGF2) and with single nucleotide polymorphisms (SNPs) for the gene maternally expressed 3 (MEG3). The cloned boars had high production of semen and were nearly equal in level of fertility to conventional pigs; they showed similar characteristics as naturally bred boars of the same strains. The expression of IGF2 was partially disturbed, but this disturbed expression was not linked to a change in developmental fate or reproductive performance. These results indicate that use of cloned boars could be highly effective for proliferation of pigs with desirable characteristics, preservation of genetic resources and risk reduction against epidemic diseases, such as foot‐and‐mouth disease, through storage of somatic cells as a precautionary measure for use in regenerating pig populations after a future pandemic.     

成体干细胞的研究现状与展望

近年来，干细胞特有的生物学特性及其潜在的生物医学应用价值使之成为生命科学领域最热点、最前沿的课题之一。随着对干细胞研究的不断深入，人们发现成体干细胞除了具有较强的自我更新、高度增殖和多向分化潜能外还具有分化方向的可塑性。作者根据国内外研究成果，综述了成体干细胞的“可塑性”研究的最新进展，以及成体干细胞可塑性研究的前景及存在的主要问题。     

弓形虫两种保种方法的试验

目的寻求一个简便、适宜的弓形虫保种方法。方法采用不同温度的冷冻保存法和细胞培养保存法对弓形虫保存进行了试验研究,为深入研究弓形虫提供保证。结果弓形虫在液氮(-196℃)、-70℃能长期保存,4℃能保存14 d。通过细胞培养能获得大量的弓形虫滋养体,对培养细胞的选择性不强。结论两种保种方法保存后弓形虫毒力不变。     

谷氨酰胺对免疫细胞的影响研究进展

谷氨酰胺作为一种条件性必需氨基酸,在动物处于特殊生长阶段如幼龄期或在应激期间都显示出明显的免疫促进作用。动物免疫力的高低直接取决于各种免疫细胞如淋巴细胞、中性粒细胞、巨噬细胞的生长情况和免疫应答能力,谷氨酰胺的免疫促进作用与影响免疫细胞生长和免疫应答能力的发挥密切相关。本文就谷氨酰胺与免疫细胞增殖、分化、凋亡、分泌和热休克反应的研究进展做一综述。     

Preliminary experiments on mechanical stretch-induced activation of skeletal muscle satellite cells in vivo

We have shown in vitro that mechanical stretch triggers activation of quiescent satellite cells of skeletal muscle to enter the cell cycle through an intracellular cascade of events including nitric oxide (NO) synthesis that results in the release of hepatocyte growth factor (HGF) from its extracellular association and its subsequent presentation to signaling receptors. In order to explore the activation mechanism in vivo, stretch experiments were conducted in the living animal using our suspension model developed. This system used the weight of the hind portion of rats to stretch the inside muscles of the left hind limb suspended for a period of 0.5–2.0 h. At the end of the stretch period, the rats received an intraperitoneal injection of bromodeoxyuridine followed by immunocytochemistry for its incorporation as an index of satellite cell activation in vivo. Depending on the period of stretch, bromodeoxyuridine labeling was increased significantly over the contralateral unstretched leg or control muscle from untreated rats. A stretched muscle extract prepared from the 2 h stretched tissue by incubating it in PBS, showed the active form of HGF as revealed by immunoblotting and it could stimulate the activation of unstretched satellite cells. Also, administering NO synthase inhibitor L‐NAME prior to muscle stretch abolished the stretch activation of satellite cells. Therefore, the results from these experiments demonstrate that stretching muscle triggers NO synthesis and HGF release, which could activate satellite cells in vivo.     

Treatment of cow-waste slurry by a microbial fuel cell and the properties of the treated slurry as a liquid manure

Resource recycling and the proper treatment of animal waste to reduce its environmental impact are currently important issues for the livestock industry. A microbial fuel cell (MFC), a new type of bioreactor, is expected to play roles in both waste‐water purification and energy recovery. However, the generation of electricity from cow waste has not yet been examined. In this study, using an MFC, we examined the possibility of generating electricity from dairy‐cow waste slurry, and analyzed the properties of the treated slurry as liquid manure for resource recycling. The MFC treatment of the slurry generated electricity in a dose‐dependent manner, and the maximum power output by the MFC from a 1 g of chemical oxygen demand/L slurry was 0.34 mW/m². After the MFC treatment, 84% of the biological oxygen demand in the slurry was removed and three essential fertilizer elements (nitrogen, phosphorus, and potassium) were retained at 84, 70, and 91% levels, respectively. The amount of ammonia nitrogen in the slurry, as an element of fast‐release fertilizer, was increased by 1.9‐fold. Although the treated slurry displayed properties that made it preferable as liquid manure, further studies to improve the electrical power output by the MFC are required for practical use.     

狗舌草提取物诱导淋巴细胞性白血病L1210细胞分化的研究

以单猪屎豆碱（MO）、槲皮素（QU）和环磷酰胺（CY）为参照，观察了狗舌草600mL／L乙醇提取物（EX）对淋巴细胞性白血病L1210细胞体外试验的形态变化；利用流式细胞术，从DNA分子水平上检查了EX对L1210细胞各周期相的影响，探讨EX对L1210细胞的分化机理。结果发现，EX能够使L1210细胞向淋巴细胞方向发展；经EX作用24h后，L1210细胞G0＋G1期的百分比较对照组明显升高。提示EX对L1210细胞增殖的抑制作用可能是由于G1期的阻滞所致。     

牛TLR4基因的遗传多态性与乳房炎的关联分析

TLR4通过识别病原体而激活免疫细胞，在先天免疫和适应性免疫防御中起着重要作用。以中国荷斯坦奶牛、三河牛和中国西门塔尔牛共397头为研究对象，利用创造酶切位点PCR法扩增243bp的目的片段，通过限制性内切酶HinfⅠ酶切来检测TLR4第3外显子的多态性，结果发现扩增产物的27bp处C到T的突变使得多态位点产生，编码的氨基酸由苏氨酸变为异亮氨酸。A、B2个等位基因在3个群体中均有分布，A等位基因占优势（大于78％），经χ^2适合性检验，三河牛在该位点未达到Hardy-Weinberg平衡状态（P〈0．05）。运用SAS8．2软件采用最小二乘法拟合线性模型，将该基因座不同基因型与奶牛乳房炎进行了关联分析，结果表明：AA基因型为乳房炎抗性基因型（P〈0．05），A等位基因为乳房炎抗性的有利基因。