Ixazomib,an oral proteasome inhibitor,exhibits potential effect in dystrophin‐deficient mdx mice

Dystrophin deficiency makes the sarcolemma fragile and susceptible to degeneration in Duchenne muscular dystrophy. The proteasome is a multimeric protease complex and is central to the regulation of cellular proteins. Previous studies have shown that proteasome inhibition improved pathological changes in mdx mice. Ixazomib is the first oral proteasome inhibitor used as a therapy in multiple myeloma. This study investigated the effects of ixazomib on the dystrophic muscle of mdx mice. MDX mice were treated with ixazomib (7.5 mg/kg/wk by gavage) or 0.2 mL of saline for 12 weeks. The Kondziela test was performed to measure muscle strength. The tibialis anterior (TA) and diaphragm (DIA) muscles were used for morphological analysis, and blood samples were collected for biochemical measurement. We observed maintenance of the muscle strength in the animals treated with ixazomib. Treatment with ixazomib had no toxic effect on the mdx mouse. The morphological analysis showed a reduction in the inflammatory area and fibres with central nuclei in the TA and DIA muscles and an increase in the number of fibres with a diameter of 20 µm² in the DIA muscle after treatment with ixazomib. There was an increase in the expression of dystrophin and utrophin in the TA and DIA muscles and a reduction in the expression of osteopontin and TGF‐β in the DIA muscle of mdx mice treated with ixazomib. Ixazomib was thus shown to increase the expression of dystrophin and utrophin associated with improved pathological and functional changes in the dystrophic muscles of mdx mice.     

Xenotransplantation of Embryonic Precursors of Human Myogenesis for the Correction of Dystrophinopathy in Mice with Hereditary Muscular Dystrophy

Human embryonic myogenic precursors were transplanted into muscles of mdx mice with hereditary dystrophin-deficient muscular dystrophy. Transplantation induced the synthesis of human dystrophin. The number of dystrophin-positive fibers progressively decreased, however, some of them were preserved even 5 months after transplantation. Our results indicate that xenogeneic transplantation of embryonic myogenic precursors compensates the genetic defect in dystrophin-deficient mice.     

骨组织工程中调节巨噬细胞行为促进骨愈合的策略

骨组织工程将材料作为骨再生支架植入缺损局部,帮助完成骨愈合。近年来随着骨免疫学的发展,人们意识到巨噬细胞的免疫应答决定着材料植入的成败。巨噬细胞具有多样性和可塑性,可以根据环境信号极化为M1样(促炎)和M2样(抑炎)表型,在骨愈合的不同阶段发挥作用。巨噬细胞的迁移、增殖、极化等行为对材料特性敏感。对目前在骨组织工程中,通过改变材料的物理特性、表面化学特性以及生物特性,设计能主动调节巨噬细胞行为的材料,从而促进骨愈合的策略进行了概括总结,相关策略包括：增加材料的粗糙度、大孔结合纳米结构、合适的电信号或机械信号、中性或阴离子表面、增加亲水性、利用免疫调节材料或向局部输送免疫活性物质等,以期为设计具有良好免疫调节性的骨组织工程材料提供思路。     

骨髓间充质干细胞移植后在mdx鼠体内的分布研究

目的： 研究骨髓间质干细胞移植治疗Duchenne型肌营养不良鼠(mdx鼠)后体内各主要器官的分布状况。方法: 取第5代SD大鼠骨髓间质干细胞，应用［3H］－TdR标记后以2×107cells／只细胞尾静脉移植到经7Gy γ射线预处理后的7-9周龄mdx鼠体内，分别于移植后24 h、48 h、4周、8周和16周测定血液、肺、肝、骨髓、骨骼肌及心肌的放射性分布计数。结果: 经7Gy γ射线放疗预处理的mdx鼠，在尾静脉移植骨髓干细胞后，24 h肺的放射性分布计数最高，为36.70±3.04；48 h肝的放射性分布计数最高，为36.74±3.28；骨髓分布计数随移植时间延长增多，2周时达到高峰，计数为38.43±4.99，以后逐渐下降，但4月时仍明显高于其它组织器官，计数为13.45±1.37；骨骼肌、心肌的放射性分布计数随移植时间延长明显增高，16周时分别达到4.79±0.94和9.55±1.53。结论: 在MSCs移植后早期，MSCs主要分布于肺、肝等血流丰富的器官，随移植时间延长逐渐归巢到骨髓定居，2周时达到高峰，此后逐渐向损伤器官如骨骼肌、心肌等定向迁移，并且随时间延长分布增加，从而为MSCs定居于肌组织并且分化为肌细胞提供了证据。     

骨形成蛋白介导的基因治疗研究进展

基因治疗是指通过导入基因的功能片段改善机体生理状况或者治疗疾病.利用基因治疗可在骨缺损或骨折局部释放骨形成蛋白（bone morphogenetic proteins,BMP）,并且无需异体载体,方法包括体内法和离体法.BMP基因治疗可以促进骨和软骨形成、脊柱融合、颌面骨和牙齿修复、肌腱韧带形成,此外对椎间盘退变性疾病也可采用BMP基因治疗.总之BMP基因治疗方法经济有效,是有前景的治疗手段.     

Histomorphometric Study of Alveolar Bone Healing in Rats Fed a Boron‐Deficient Diet

Bone healing after tooth extraction in rats is a suitable experimental model to study bone formation. Thus, we performed a study to determine the effects of boron (B) deficiency on bone healing by using this model. The first lower right molar of weanling Wistar rats was extracted under anesthesia. The animals were divided into two groups: +B (adequate; 3 mg B/kg diet), and ?B (boron‐deficient; 0.07 mg/kg diet). The animals in both groups were killed in groups of 10 at 7 and 14 days after surgery. The guidelines of the NIH for the care and use of laboratory animals were observed. The mandibles were resected, fixed, decalcified, and embedded in paraffin. Buccolingually oriented sections were obtained at the level of the mesial alveolus and used for histometric evaluations. Total alveolar volume (TAV) and trabecular bone volume per total volume (BV/TV) in the apical third of the alveolus were determined. Percentages of osteoblast surface (ObS), eroded surface (ES), and quiescent surface (QS) were determined. No statistical significant differences in food intake and body weight were observed. Histomorphometric evaluation found ?B rats had 36% and 63% reductions in BV/TV at 7 and 14 days, respectively. When compared with +B rats, ?B rats had significant reductions (57% and 87%) in ObS concomitantly with increases (120% and 126%) in QS at 7 and 14 days, respectively. The findings show that boron deficiency results in altered bone healing because of a marked reduction in osteogenesis. Anat Rec, 291:441–447, 2008. © 2008 Wiley‐Liss, Inc.     

Morpholino-induced exon skipping stimulates cell-mediated and humoral responses to dystrophin in mdx mice

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded ‘revertant’ dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

聚-DL-乳酸膜引导兔桡骨缺损再生的实验研究

本实验旨在观察可降解聚 - DL-乳酸膜引导兔桡骨缺损再生的现象 ,并探讨其作用和机制。取 4 0只成年新西兰大白兔建立双侧桡骨缺损模型。左侧为实验侧 ,以聚 - DL-乳酸膜卷成管状桥接骨缺损 ;右侧为对照侧 ,桡骨缺损不做处理。术后 2、4、8、12周分别处死动物 ,行大体观察、X线摄片、组织学观察和骨生物力学检测。实验侧术后 2周 ,可见隔膜管两端已为软组织覆盖 ,膜管外两端可见多量新生骨痂形成 ,膜管内骨断端间充满与膜管形状相适应的血肿和纤维骨痂。术后 12周膜管颜色明显变白 ,仍然保持原有外形无塌陷 ,膜管外骨痂已基本消失 ,膜管内骨缺损均已骨性愈合。对照侧术后 2周骨缺损区被大量的结缔组织充填 ,12周时无 1例愈合 ,骨断端均已封闭而形成典型的骨不连。对照侧均不能进行力学测试 ,实验侧 12周组新生骨的生物力学指标明显优于 8周组 (P<0 .0 5 )。因此 ,聚 - DL-乳酸膜能够成功引导骨再生 ,可以用于临床治疗骨缺损和骨不连。     

利用壁厚分析法定量判断胫骨骨折骨愈合程度

目的 利用壁厚分析法定量判断胫骨骨折愈合程度,为临床判断胫骨骨不连和骨延迟愈合提供直观的诊断依据.方法 对48例患者下肢患侧与健侧进行三维建模后,计算最大壁厚(maximum wall thickness,MWT),并进行比值计算,将其对比值(B值)作为骨愈合程度的量化指标全程动态观察.当BMWT2＞0.9且BMWT1...     

Hereditary muscular dystrophy in mdx mice as a homologous model for introduction of cell technologies in the treatment of progressive muscular dystrophies in humans

Life-time monitoring of the main clinical and laboratory manifestations of hereditary muscular dystrophy in mdx mice confirmed the presence of mutation in exon 23 of dystrophin gene and the absence of this protein in skeletal muscles of mutant animals. Muscular dystrophy in mice was similar to human progressive muscle disorder, which allows the use of this model for the development of cell technologies for the treatment of hereditary muscular diseases in humans.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 10, pp. 477–480, October, 2004     

Hibernating Little Pocket Mice Show Few Seasonal Changes in Bone Properties

Periods of disuse or physical inactivity increases bone porosity and decreases bone mineral density, resulting in a loss of bone mechanical competence in many animals. Although large hibernators like bears and marmots prevent bone loss during hibernation, despite long periods of physical inactivity, some small hibernators do lose bone during hibernation. Little pocket mice (Perognathus longimembris) remain underground during winter hibernation and undergo bouts of torpor and interbout arousals, but the torpor bout duration is shorter than other rodent hibernators. Additionally, little pocket mice may enter torpor during summer estivation. In this study, cortical and trabecular bone architectural, mineral, and mechanical properties were analyzed for femurs from little pocket mice captured during 8 different months (March to October) to determine seasonal effects on bone. There were no differences in any bone properties between the pre‐hibernation month of October and the post‐hibernation month of March, suggesting winter hibernation did not adversely affect bone properties. However, cortical area was higher in March than April, May, and June. Bone mechanical and osteocyte lacunar properties were not different between any months. Trabecular bone in the distal femoral epiphysis showed no changes between months. The distal femoral metaphyseal region showed higher trabecular spacing and lower trabecular number in May than August, otherwise, there were no differences in trabecular parameters. The few monthly differences in bone properties may be due to physical inactivity from periodic summer estivation or from the timing of birth and growth in spring and summer months. Anat Rec, 300:2175–2183, 2017. © 2017 Wiley Periodicals, Inc.     

Effective detection of corrected dystrophin loci in mdx mouse myogenic precursors

Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction.     

Aquaporin 4 Expression in the mdx Mouse Diaphragm

Expression of aquaporin (AQP) 4 in the surface membranes of skeletal myofibers is well established; however, its functional significance is still unknown. The alterations of AQP4 expressions in dystrophic muscles at RNA and protein levels have been reported in various dystrophic muscles such as dystrophinopathy, dysferlinopathy, and sarcoglycanopathy. We are interested in the relationship between the severity of dystrophic muscle degeneration and the expression of AQP4. Here we compared the AQP4 expression of the limb muscles with that of diaphragms in both mdx and control mice. The dystrophic muscle degeneration, such as rounding profile of cross sectional myofiber shape, dense eosin staining, central nuclei, and endomysial fibrosis in mdx mice, were more marked in diaphragms than in limb muscles. The decrease of AQP4 expression at protein level was more marked in diaphragms than in the limb muscles of mdx mice. However, the expression of AQP4 mRNA in the diaphragms of mdx mice was not reduced in comparison with limb muscles of mdx mice. The present study revealed that AQP4 expression at protein level was correlated with the severity of dystrophic changes in muscle tissues of mdx mice.     

转基因修饰后的自体骨髓间充质干细胞移植治疗mdx鼠后成肌调节因子的表达研究

目的探讨携带micro-dystrophin基因的自体骨髓间充质干细胞移植入mdx鼠后在移植鼠体内分化为肌细胞的可能机制。方法采用逆转录病毒介导micro-dystrophin基因转染mdx小鼠MSCs（mMSCs）,通过尾静脉注射移植治疗mdx鼠,在移植后免疫荧光检测micro-dystrophin的表达并在不同时间点检测MyoD的表达。结果移植后成功检测到micro-dystrophin,其表达随着移植时间的延长而增加;随移植时间延长MyoD阳性肌纤维比例增加,分别达到9%（4周时）、15%（8周时）、28%（12周时）。RT-PCR和Westernblot也发现,随着移植时间的延长,MyoD表达增加。结论自体mMSCs可携带外源性micro-dystrophin基因在受体鼠体内分化为micro-dystrophin阳性肌细胞,移植入的干细胞向肌细胞的分化是一个持久的、连续的过程,成肌调节因子在调节其分化过程中发挥了重大作用。     

Mechanical and morphological aspects of the calcaneal tendon of mdx mice at 21 days of age

A relationship between compromised muscles and other tissues has been demonstrated in mdx mouse, an animal model studied for understanding of Duchenne muscular dystrophy. The hypothesis is that changes in the calcaneal tendon of mdx mice occur previous to the onset of rigorous and most marked episodes of muscle degeneration, which start suddenly after 21 days of life. Thus, this study aimed to identify possible alterations in the calcaneal tendon of mdx mouse at 21 days of age. Control and mdx tendons were submitted to mechanical tensile testing, quantification of hydroxyproline, and staining with toluidine blue and picrosirius red. Hydroxyproline content was similar between mdx and control groups. The control tendon presented higher mechanical strength (load, stress, and elastic modulus) and its morphological analysis showed a larger number of round fibroblasts, nuclei with well‐decondensed chromatin, and slightly metachromatic well‐stained cytoplasmic material, different from that observed in mdx tendons. The results suggest that the absence of dystrophin in mdx mouse can provoke directly or indirectly alterations in the mechanical properties and morphology of the calcaneal tendon. Anat Rec, 296:1546–1551, 2013. © 2013 Wiley Periodicals, Inc.     

接骨板不同螺钉布局下骨愈合过程有限元分析

目的探究螺钉布局方式对骨愈合的影响,为相关的骨折治疗提供基础。方法采用有限元方法研究不同螺钉布局方式下骨愈合过程;采用骨折块应变理论(interfragmentary strain theory,IFS)模拟骨痂的生长变化过程;利用Python语言对ABAQUS进行二次开发,使得迭代过程中骨痂所划分的每个单元都根据IFS理论更新弹性模量,从而模拟骨折愈合过程。结果不同数量螺钉对骨愈合过程的影响小于不同接骨板工作长度对骨愈合过程的影响;稳定固定的前提下,接骨板工作长度一定时,螺钉数量变化对接骨板或螺钉的应力影响较小;接骨板工作长度不同,对接骨板及螺钉的应力影响较大,且工作长度变化对螺钉上应力变化的影响大于对接骨板的影响。结论相较于螺钉数量,对于接骨板工作的长度应着重考虑,应当遵循在保证内固定系统稳定的前提下,减少接骨板上的螺钉数量,并对接骨板工作长度的选择进行恰当的选择。     

Restoration of gastrocnemius muscle in mdx mice of different age after injury and implantation of xenogenic muscle tissue

The intensity of regeneration of crossed gastrocnemius muscle was evaluated in two groups of mdx mice of different age 2 weeks after implantation of crushed muscle tissue from newborn rats into the wound defect area. The effect of xenoplasty manifested in increased weight of the damaged muscle. The effect was observed in mice aging 12–16 weeks but not in those aged 40–48-weeks. Structural changes in the skeletal muscle tissue intrinsic of mdx mice and augmenting with age were detected in intact mice before the experiment. Activity of muscle fiber regeneration in intact and injured muscle of 40–48-week-old mice was significantly lower than in 12–16-week-old ones. Myoblasts of the xenogenic transplant retained viability in recipient muscles for at least 2 weeks. Post-traumatic regeneration was stimulated in only 12–16-week animals. Xenoplasty was ineffective in older animals and even somewhat enhanced the destructive processes in the muscle. It seems that age-specific regeneration activity of the recipient skeletal muscle tissue should be taken into consideration in the development of effective strategy of cell therapy for progressive muscular dystrophy. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 8, pp. 216–220, August, 2006     

Therapeutic effects of exon skipping and losartan on skeletal muscle of mdx mice

Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor–beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.