利用单根肌纤维法分离和培养猪骨骼肌卫星细胞及其成肌诱导分化

建立单根肌纤维法体外培养猪骨骼肌卫星细胞的体系,了解其增殖和成肌特性。通过Ⅰ型胶原酶消化,从猪骨骼肌中分离完整的单根肌纤维并培养,用细胞免疫荧光鉴定肌纤维上的卫星细胞,随后对从单根肌纤维上游离出来的卫星细胞进行细胞免疫荧光染色,传代培养,成肌诱导分化和Western blot分析骨骼肌卫星细胞成肌特异性蛋白的表达。结果显示:分离并培养的单根肌纤维上附着有卵圆形的细胞,并随时间的推移,细胞缓慢向外迁移并增殖,卫星细胞特异性标志基因对盒转录因子(Paired protein box,Pax7)和成肌分化抗原(Myogenic Differentiation Antigen,MyoD)免疫荧光染色呈阳性,且阳性率达到90%以上。成肌诱导分化后,细胞开始汇合,并呈方向性生长,最终形成多核肌管,且成肌特异性标志基因Myogenin和myosin heavy chain(MyHC)表达呈阳性。MyoD蛋白高表达于增殖期,而Myogenin和MyHC在进入分化期才表达。该实验成功建立了猪骨骼肌单根肌纤维的体外培养方法并获得了高纯度的卫星细胞,为骨骼肌卫星细胞进行活体移植治疗相关疾病研究提供了实验材料。     

金鳟骨骼系统解剖观察

采用常规煮沸剥离方法对金鳟骨骼系统的组成进行观察,得到了金鳟的完整骨骼标本和单个骨骼标本。结果表明：金鳟由353块骨骼组成,其中主轴骨骼277块,附肢骨骼76块。筛骨区发达,其中围眶骨6对,各围眶骨中皆有感觉管穿过。牙齿在口咽腔中分布很广,捕食能力强。金鳟的躯干部由150块骨骼组成,体型侧扁,背鳍基部短,在背鳍之后还有1个小脂鳍。附肢骨骼中无坚硬的鳍棘出现。附肢骨骼中的腰带,构造非常简单,仅为1块三角形骨板,起到承重的功能。     

利用iTRAQ蛋白质组技术筛选与鸡喙畸形相关的候选蛋白

【目的】中国地方鸡种如北京油鸡、清远麻鸡存在喙畸形现象,表现为上下喙咬合不全,呈交叉状。严重影响鸡饮水和采食,从而影响个体发育和生产性能的发挥,造成一定经济损失。笔者根据喙畸形个体的系谱记录,发现喙畸形的形成受到遗传因素的影响,但具体机制尚不明确。蛋白是行使各种生物学功能的最终形式之一,喙畸形个体的发生可能是由于核心蛋白或者相关调控蛋白代谢异常造成的。利用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation,iTRAQ）技术以及生物信息学分析方法,筛选鸡畸形喙与正常喙中差异表达的蛋白,作为喙畸形相关的重要候选蛋白,为进一步研究北京油鸡喙畸形的遗传机制奠定基础。【方法】挑选3只120日龄喙畸形的公鸡作为试验组,编号为W1、W2、W3,同时,挑选与试验组个体为同胞关系（全同胞或半同胞）的喙正常公鸡作为对照组,编号为Z1、Z2、Z3。将喙畸形与其同胞正常个体作为一个比对组,iTRAQ试验具体包含3个比对组,即W1 vs Z1,W2 vs Z2,W3 vs Z3。屠宰个体后,剔除喙组织周围肌肉和筋膜,分离得到喙上颌骨和下颌骨,提取总蛋白样品,应用6个 iTRAQ标签标记各蛋白样品,经过色谱层析预分离,联合液相串联质谱分析,采用Mascot 2.3.02软件对蛋白进行鉴定和定量分析。试验组（W）与其同胞对照组(Z)样本比较（W1 vs Z1,W2 vs Z2,W3 vs Z3）,选择肽段数≥2,表达差异值＞1.2（上调）或＜0.83（下调）,且P＜0.05的蛋白作为差异表达蛋白。【结果】利用iTRAQ技术一共在喙组织中鉴定到3 372个蛋白,鉴定到的特异性肽段有12 769个。其中原鸡蛋白1 869个,分子质量主要分布在10-100 kD之间。3个比对组共鉴定出159个表达量有显著差异的蛋白质,其中包含70个表达上调的蛋白,89个表达下调的蛋白。统计各比对组蛋白表达差异值发现,表达量差异较大的上调蛋白质有LPL、MLC-2、CO9A1、MATN3、HSP90B1等,表达量差异较大的下调蛋白质有MBP、RLA1、PRVM、HAPLN1等。结合已经报道的这些蛋白的生物学功能,其中CO9A1、MATN3、HAPLN1与软骨合成和软骨骨化相关,CO9A1是带状软骨纤维的组成物质,MATN家族是非胶原性细胞外基质蛋白家族,HAPLN1是软骨细胞外基质的重要组成成分;PRVM是细胞内钙离子结合蛋白,参与调控Ca2+离子信号通路;LPL是多功能酶,主要在脂质代谢和转运过程中起作用,参与调控PPAR信号通路。初步筛选出CO9A1、MATN3、HAPLN1、PRVM、LPL作为与鸡喙畸形相关的候选蛋白。【结论】差异表达蛋白的发现为鸡喙畸形形态的发生提供蛋白质水平上的理论依据。     

CORRECTION OF ANGULAR DEFORMITY BY ASYMMETRIC PHYSEAL GROWTH AND BONE REMODELLING

Angular deformities of 25 or 37 degrees valgus were created by transverse wedge osteotomies in the diaphysis of the radius in 3-month-old Cocker Spaniels. Spontaneous correction was judged from radiographs until skeletal maturity. The magnitude of correction was estimated, using the opposite normal leg as a control. Asymmetrical physeal growth corrected the induced angulation between the proximal and distal radial joint plane surfaces (joint plane angle). The average correction of the joint plane angle was 80% in the 25 degree group, and 72% in the 37 degree group. Bone remodeling of the radial shaft corrected the induced diaphyseal angulation by 50% in both groups during the study.     

Spinal deformity in triploid grass carp Ctenopharyngodon idella (Valenciennes)

From mid-2004 to mid-2005, several grass carp, Ctenophayngodon idella (Valenciennes), showing evidence of spinal deformity were presented to the Aquatic Animal Health Program, Cornell University. The carp were from three separate locations in New York State. The first case involved several fish from a natural body of water in the Catskill Mountain region of south-eastern New York State. The second was a single affected individual from a private pond in the Fingerlakes region of Central New York State. The third was a single individual from the Cold Springs Harbor Fish Hatchery, Cold Springs Harbor, Long Island. All fish were at least 7 years of age. Radiographs and computed tomography (CT) scans revealed the deformities to be of bony origin. The spinal deformities were characterized by variable amounts of kyphosis, scoliosis and rotation. While it is not possible to determine the specific cause(s) of the lesions, we consider a genetic component as a likely contributor to the observed pathology.     

3-甲基-4-硝基酚对雄性小鼠生殖毒性的研究

目的探讨3-甲基-4-硝基酚(PNMC)对雄性小鼠的生殖毒性作用。方法 20只小鼠随机分成阴性对照组和PNMC组(分0.9、9和90mg/kg·bw组),经口灌胃染毒30天,观察睾丸和精子的形态变化。结果染毒组小鼠各剂量睾丸生精细胞发生坏死,曲细精管腔内出现细胞团,精子细胞分化异常,形成均质红染颗粒,睾丸曲细精管生精上皮/直径比值在90mg/kg组明显高于对照组;染毒组小鼠精子畸形率明显高于对照组(P<0.05),并且随着染毒浓度的增加,畸形率有增加的趋势。结论 3-甲基-4-硝基酚不仅可以引起生精细胞坏死和抑制精子形成,还可以诱导精子畸变,从而对生殖系统造成损伤。     

Use of hinged circular fixator constructs for the correction of crural deformities in three dogs

Background Hinged circular external skeletal fixator constructs are used to perform sequential correction of angular limb deformities, often with resultant limb segment lengthening, via distraction osteogenesis. Although there are several reports describing the use of these constructs for correction of antebrachial deformities in dogs, there is little information regarding their use on other limb segments. This report describes the use of hinged circular fixator constructs for the correction of acquired crural deformities in three skeletally immature dogs. Case reports Two dogs had purely frontal plane deformities (one valgus, one varus) and the third dog had frontal (valgus) and sagittal (recurvatum) components to its deformity. At the time of long‐term evaluation, frontal plane angulation relative to the contralateral limb improved from 40° to 22° of valgus, 30° to 5° of valgus, and 20° to 1° of varus in the three individual dogs. Tibial length discrepancies of 12% and 22% that were initially present in two dogs were improved to 6% and 10%, respectively, of the contralateral tibial length at the time of final evaluation; both dogs had compensatory growth of the ipsilateral femur and all dogs had an excellent functional outcome. Conclusion These cases illustrate the value of using hinged circular fixator constructs for correction of crural angular deformities, particularly when length discrepancies of the tibia are present.     

Skeletal deformities associated with nutritional congenital rickets in newborn lambs

Abstract

CASE HISTORY: A group of 545 pregnant rising 2-year-old Coopdale ewes on a Southland sheep farm were grazed over winter on a fodder beet (Beta vulgaris) crop. Subsequently, 45 out of approximately 750 lambs were born with a variety of skeletal deformities, including shortened limbs, varus and valgus angular limb deformities, palmar grade stance and cranial bowing of the carpus. Analysis of the crop showed the fodder beet contained a low percentage of phosphorus. In addition, 60 out of 460 rising 2-year-old ewes that had been grazed on the fodder beet crop as 1-year-olds had incisor abnormalities and malocclusion.

PATHOLOGICAL FINDINGS: Two affected lambs (1-day-old and 3-days-old) with representative clinical signs examined postmortem were found to have markedly enlarged costochondral junctions, and noticeably enlarged long bone metaphyses. In addition, one lamb had a dense band of metaphyseal sclerosis beneath the physes of all long bones examined. Histopathological findings included small islands and columns of chondrocytes and eosinophilic cartilage matrix present in the metaphysis. Metaphyseal trabeculae were disorganised and often lined by accumulations of pale pink osteoid; similar pale pink osteoid was also present in the cortices. Unerupted molar teeth in the affected lambs lacked a layer of enamel, and the dentine was irregular with globular basophilia.

DIAGNOSIS: The gross and histopathological lesions were consistent with a diagnosis of rickets.

CLINICAL RELEVANCE: Nutritional congenital rickets has not been previously diagnosed in sheep, but is a recognised disease of human infants with vitamin D deficient mothers. The rickets in affected lambs was most likely associated with phosphorus deficiency as a result of the pregnant ewes grazing fodder beet during gestation. While vitamin D deficiency was not definitively ruled out in these cases, practitioners are alerted to the possible effects of feeding phosphorus-deficient fodder beet to ewes for long periods during gestation and to 1-year-old sheep during important growth periods.     

Unusual congenital deformities of the lower limb in two calves

Congenital deformities of the lower limb were observed in 2 calves from different properties. One was a newborn Holstein-Friesian bull calf with polydactyly and polypodia of the right fore-limb.The other was an 8-month-old Friesian/Jersey crossbred heifer calf, which had a mirror image duplication of the plantar/palmar half of the distal portion of the digits. The abnormalities in the second calf have not previously been described in cattle. This paper presents details of the clinical, radiological and necropsy findings in these 2 calves.