New Insights into the Neural Differentiation Potential of Canine Adipose Tissue‐Derived Mesenchymal Stem Cells

Adipose tissue‐derived stem cells (ASCs) can be obtained from different adipose tissue sources within the body. It is an abundant cell pool, easily accessible, suitable for cultivation and expansion in vitro and preparation for therapeutic approaches. Amongst these therapeutic approaches are tissue engineering and nervous system disorders such as spinal cord injuries. For such treatment, ASCs have to be reliably differentiated in to the neuronal direction. Therefore, we investigated the neural differentiation potential of ASCs using protocols with neurogenic inductors such as valproic acid and forskolin, while dog brain tissue served as control. Morphological changes could already be noticed 1 h after neuronal induction. Gene expression analysis revealed that the neuronal markers nestin and βIII‐tubulin as well as MAP2 were expressed after induction of neuronal differentiation. Additionally, the expression of the neurotrophic factors NGF, BDNF and GDNF was determined. Some of the neuronal markers and neurotrophic factors were already expressed in undifferentiated cells. Our findings point out that ASCs can reliably be differentiated into the neuronal lineage; therefore, these cells are a suitable cell source for cell transplantation in disorders of the central nervous system. Follow‐up studies would show the clinical benefit of these cells after transplantation.     

Molecular and Cellular Characterization of Buffalo Bone Marrow‐Derived Mesenchymal Stem Cells

Immune privileged mesenchymal stem cells (MSCs) can differentiate into multiple cell types and possess great potential for human and veterinary regenerative therapies. This study was designed with an objective to isolate, expand and characterize buffalo bone marrow‐derived MSCs (BM‐MSCs) at molecular and cellular level. Buffalo BM‐MSCs were isolated by Ficoll density gradient method and cultured in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum (FBS). These cells were characterized through alkaline phosphatase (AP) staining, colony‐forming unit (CFU) assay, mRNA expression analysis (CD 73, CD 90, CD 105, Oct4 and Nanog), immunolocalization along with flow cytometry (Stro 1, CD 73, CD 105, Oct4, Sox2 and Nanog) and in situ hybridization (Oct4 and Sox2). Multilineage differentiation (osteogenic, adipogenic and chondrogenic) was induced in vitro, which was further assessed by specific staining. Buffalo BM‐MSCs have the capacity to form plastic adherent clusters of fibroblast‐like cells and were successfully maintained up to 16^th passage. These cells were AP positive, and further CFU assay confirmed their clonogenic property. RT‐PCR analysis and protein localization study showed that buffalo BM‐MSCs are positive for various cell surface markers and pluripotency markers. Cytoplasmic distribution of mRNA for pluripotency markers in buffalo BM‐MSCs and multilineage differentiation were induced in vitro, which was further assessed by specific staining. To the best of our knowledge, this is the first report of buffalo BM‐MSCs, which suggests that MSCs can be derived and expanded from buffalo bone marrow and can be used after characterization as a novel agent for regenerative therapy.     

绵羊羊水干细胞外源基因高效转染及快速筛选方法的建立

试验旨在建立绵羊羊水来源多潜能干细胞(amniotic fluid-derived stem cells,AFSC)外源基因高效转染及快速筛选方法,同时保持其多潜能特性。采用脂质体法,以pCMV-DsRed为报告质粒,转染绵羊AFSC,36 h后,流式细胞仪分析外源基因的瞬时转染效率为69.17%。以浓度为6 mg/mL的G418筛选5 h获得纯化转基因绵羊AFSC单克隆。转基因绵羊AFSC RT-PCR检测表达Oct-4、SSEA-1,体外悬浮培养可形成类胚体。结果表明,转基因标记绵羊AFSC保持了发育全能性的潜能,构建的转基因绵羊AFSC可以示踪性研究其在体内的分化路径和最终宿主。     

蒙古羊羊水源干细胞分离培养及其成骨分化研究

试验旨在建立羊水源干细胞系并对其诱导形成成骨细胞的潜能进行研究。在胎牛血清浓度为15%的条件下对羊水源干细胞进行建系,并检测其类胚体形成能力;检测不同代数细胞的干细胞标志基因Oct-4、SH2、SSEA-1、Nanog、HLA-ABC、CD117的表达情况;利用地塞米松、β-甘油磷酸钠、抗坏血酸等因子进行诱导。结果表明,羊水源干细胞在体外可以持续增殖,悬浮培养形成的类胚体表达三胚层相关标记基因,经诱导可形成成骨细胞。试验成功分离了蒙古羊的羊水源干细胞并建系,且其具有向成骨细胞分化的潜能。     

蒙古马骨髓间充质干细胞的分离培养及多向分化潜能的研究

旨在探索一个高效分离和扩增蒙古马骨髓间充质干细胞的方法,并通过形态学和多向分化能力进行鉴定.抽取蒙古马骨髓标本,利用密度梯度离心和差异贴壁法分离MSCs,体外扩增后传代,进行相差显微镜形态学观察.在地塞米松、IBMX、胰岛素及罗格列酮的作用下向脂肪细胞诱导分化,以及在地塞米松、VitC、β-磷酸甘油等作用下向成骨细胞诱导分化.结果显示原代和传代细胞呈梭形成纤维细胞样外观,生长增殖能力良好.经定向诱导分化后,细胞分别呈现脂肪细胞、成骨细胞的表型特征.证明所纯化的细胞为尚未分化的基质细胞,而不是组织的成体细胞,它具有多向分化潜能.     

Long‐Term Management with Adipose Tissue‐Derived Mesenchymal Stem Cells and Conventional Treatment in a Dog with Hepatocutaneous Syndrome

Hepatocutaneous syndrome (HS) is an uncommon skin disorder that occurs in conjunction with liver disease and is diagnosed based on decreased plasma concentrations of amino acids and the histopathology of skin lesions. The survival period generally is <6 months. A 10‐year‐old castrated male Maltese dog was presented for evaluation of lethargy, polyuria, polydipsia, and skin lesions including alopecia, erythema, and crusts. Based on increased liver enzyme activity, low plasma amino acid concentrations, and findings from liver cytology and skin biopsy, the dog was diagnosed with HS. In addition to administration of antioxidants, hepatoprotective agents, and amino acids IV, allogenic adipose tissue‐derived mesenchymal stem cells were infused 46 times over a 30‐month period: 8 times directly into the liver parenchyma guided by ultrasonography and the remainder of the times into peripheral veins. After commencing stem cell therapy, the dog's hair re‐grew and the skin lesions disappeared or became smaller. During ongoing management, the patient suddenly presented with anorexia and uncontrolled vomiting, and severe azotemia was observed. The dog died despite intensive care. On necropsy, severe liver fibrosis and superficial necrolytic dermatitis were observed. The dog survived for 32 months after diagnosis. A combination of amino acid and stem cell therapy may be beneficial for patients with HS.     

盘羊脐带间充质干细胞的分离培养及诱导分化

试验旨在研究野生盘羊脐带间充质干细胞（umbilical cord mesenchymal stem cells,UC-MSCs）的分离、培养及体外多向分化潜能等生物学特性。取盘羊分娩后的新生雄性胎儿脐带,采用组织块培养法分离盘羊UC-MSCs后进行体外培养,并对UC-MSCs进行成骨、成软骨和成脂诱导分化,检测其多向分化潜能。结果显示,应用组织块培养法获得的盘羊UC-MSCs具有UC-MSCs特有的呈簇、成纤维状的特性,细胞呈"S"型曲线生长,细胞的倍增时间平均为33.50 h,且可以向成骨细胞、成软骨细胞和成脂细胞分化。诱导分化特异性染色结果表明,野生盘羊UC-MSCs经诱导后的成骨细胞经茜素红S染色呈现典型的橙红色钙沉积物聚集,成软骨细胞经阿利新蓝染色呈现蓝色的软骨基质,成脂细胞经油红O染色呈现深红色的脂滴。实时荧光定量PCR检测结果发现,随着诱导时间的延长,成骨分化的细胞中骨内γ-羧基谷氨酸蛋白（BGLAP）、骨桥蛋白基因（OPN）、Runt相关转录因子2（RUNX2）基因的相对表达量极显著升高（P<0.01）;成软骨分化的细胞中光蛋白聚糖（LUM）基因的表达量明显增加,而双糖链蛋白多糖（BGN）和Y染色体性别决定区基因盒9（SOX9）基因表达量极显著提高（P<0.01）;成脂分化的细胞中脂蛋白酶（LPL）和过氧化物酶体增殖因子活化受体γ（PPAR-γ）基因的相对表达量极显著上升（P<0.01）。试验结果表明,用盘羊胎盘附带的脐带组织可以分离到盘羊UC-MSCs,且分离到的UC-MSCs具有分化为成骨细胞、成软骨细胞及成脂细胞等多向分化潜能。     

北京油鸡角膜缘干细胞分离培养及分化潜能研究

【目的】 研究北京油鸡角膜缘干细胞（LSCs）分离、培养、鉴定及多向分化潜能等生物学特性。【方法】 取10胚龄健康北京油鸡鸡胚的角膜缘,采用分散酶Ⅱ（dispaseⅡ）联合胰蛋白酶二步法分离细胞,进行体外培养,绘制生长曲线,通过免疫荧光和RT-PCR技术鉴定LSCs特异性标志物细胞角蛋白3（CK3）、CK12、细胞周期调控蛋白p63、ATP结合盒转运蛋白（ABCG2）和细胞角蛋白19（CK19）的表达;通过成骨和成软骨诱导检测其多向分化潜能。【结果】 获得的北京油鸡细胞折光性强、贴壁后呈多角形,生长曲线呈典型的S型,群体倍增时间（PDT）随着代次的升高逐渐下降;免疫荧光检测结果表明,分离的细胞中ABCG2、p63和CK19均呈阳性表达,CK3和CK12不表达;RT-PCR检测结果表明,分离的细胞表达p63、CK19、ABCG2,不表达CD45,说明分离的细胞为LSCs。LCSs成骨诱导分化后,可见被茜素红染色的矿化钙结节,RT-PCR检测结果表明其表达成骨关键基因骨桥蛋白（OPN）和Ⅰ型胶原蛋白（Col-Ⅰ）;成软骨诱导分化后,可见被阿利新蓝染色的软骨组织,RT-PCR检测结果表明其表达成软骨细胞关键基因转录因子性别决定区Y框9（SOX-9）和Ⅱ型胶原蛋白（Col-Ⅱ）。【结论】 本研究成功从10胚龄北京油鸡胚胎角膜缘中分离LSCs,且其具有良好的增殖活力及成骨和成软骨能力,结果可为禽类LSCs的资源保存研究提供参考。     

Serum Levels of Cardiac Markers NT‐proANP and NT‐proBNP in Brachycephalic bitches at Different Gestational Stages

The aim of this study was to determine serum levels of natriuretic peptide precursors (NT‐proANP and NT‐proBNP) during pregnancy in brachycephalic bitches. Fifteen healthy multiparous bitches were selected for this prospective study. Serum levels of NT‐proANP and NT‐proBNP were measured during anoestrous and at 14, 35, 42, 49 and 56 days (2nd, 5th, 6th, 7th and 8th weeks) of pregnancy. Fourteen animals had normal gestations, and one bitch developed single foetus syndrome. The natriuretic peptide levels of this animal were not included in this study; however, it is important to report that its NT‐proANP levels were four times greater than those of normal patients. There was no significant difference (p = 0.072) in NT‐proBNP levels between anoestrous (0.20 ± 0.10 ng/ml) and the different pregnancy weeks (0.27 ± 0.12 ng/ml). There was a positive correlation (p < 0.0001) between NT‐proANP and gestational age, and the levels of this marker increased significantly (p < 0.0001) during the 6th (0.26 ± 0.06 ng/ml), 7th (0.28 ± 0.04 ng/ml) and 8th weeks (0.29 ± 0.05 ng/ml) when compared to anoestrous (0.18 ± 0.02 ng/ml). NT‐proANP serum levels are correlated with gestational development and may be indicative of cardiovascular adaptation in canine brachycephalic pregnancy.     

Molecular Diversity of Campylobacter coli and C. jejuni Isolated from Pigs at Slaughter by flaA‐RFLP Analysis and Ribotyping

A population of porcine isolates of Camplobacter jejuni (n=11) and C. coli (n=17) were examined for genotypic relatedness employing ribotyping, as well as polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of the flagellin (fla)A gene locus. PCR was employed to amplify a 533 bp fragment from the flaA gene, including the previously described short variable region (SVR), employing the novel primers, A2 and A1 and successfully generated this amplicon for all wild‐type strains examined (n=28) of both C. jejuni and C. coli, as well as with both type strains, i.e. C. jejuni NCTC 11351 and C. coli NCTC 11366. Individual genotypes were assigned to each isolate typed employing the four typing methods (flaA‐RFLP_{Hae III}, flaA‐RFLP_{Pst I} ribotyping_{Hae III} and ribotyping_{Pst I},) and were assigned an arbitrary genotype code in ascending alphabetical order in comparison with a database of established genotypes for each of the methods employed. This study showed that several flaA‐RFLP and ribopatterns existed within C. jejuni and C. coli, and demonstrated a heterogeneous diversity of strains occurring in the pigs examined. Ribotyping of strains with 16S and 23S rRNA with Pst I and Hae III digested chromosomal DNA allowed subdivision of strains into nine and eight groups, respectively. RFLP analyses with Pst I and Hae III digests probed with the flaA gene probe allowed subdivision of strains into eight and eleven subtypes, respectively. Employment of RFLP with the flaA nucleic acid probe and Hae III digests produced the greatest amount of variation of any genotyping scheme employed. Although there was a high degree of variability demonstrated by both typing methods, most isolates (>60%) clustered into four main genotypes, i.e. genotypes A–D. FlaA‐PCR–RFLP typing demonstrated that the majority of isolates, 67.9 and 60.7%, were included in these four main genotypes for Pst I and Hae III restriction digests, respectively, although there was a high prevalence (7/11; 63.6%) of fla_{Hae III} genotype A occurring within the C. jejuni isolates. Likewise, ribotyping studies demonstrated that most isolates were clustered into these four main genotypes, accounting for 81.5 and 60.7% of isolates for Pst I and Hae III restriction digests, respectively. This may indicate that the clonal population of campylobacters within this pig population is largely composed of persistent and dominant types, with a smaller number of hypervariable subtypes. Such data may useful in determining epidemiological routes of transmission of campylobacters from animal to animal, as well as helping to identify virulence determinants in persistent subtype populations.     

TIMP3基因在长白猪和通城猪不同组织及不同时期骨骼肌中的表达分析

采用定量PCR(Quantification PCR, QPCR)方法,比较分析了基质金属蛋白酶3(TIMP3)基因在长白猪和通城猪间的组织表达谱,同时比较分析了其在这2个猪种胚胎期(33, 45, 55, 65, 70和90 d)和出生后(0, 9, 30, 60, 120和160 d)骨骼肌生长发育过程中的差异性表达。组织表达谱结果表明,TIMP3基因在不同猪种的各组织器官中广泛表达,除在腿肌和胰腺中有所差异外,TIMP3基因在两个猪种间的组织表达谱基本是一致的,在肺脏和子宫中表达最高,在背肌、脾脏和胃中表达较高,在肠、心脏、肾脏和肝脏中表达最低。与出生后阶段相比,TIMP3基因在2个猪种胚胎期均显著高表达(P＜0.05),但其表达变化趋势在2个猪种间存在差异;与在长白猪胚胎期相比,该基因在通城猪胚胎期中的高表达水平维持在一个更大的范围内。本试验结果提示,TIMP3基因可能参与不同类型猪种骨骼肌生长发育异步性的调控,影响到不同类型猪种的产肉性状。     

THBS3基因在长白猪和通城猪不同组织和不同时期骨骼肌中的表达分析

本研究主要探究血小板反应蛋白3 (thrombospondin-3,THBS3)基因在不同组织及不同时期骨骼肌生长发育过程中的表达规律。利用实时荧光定量PCR方法对THBS3基因在长白猪和通城猪中的组织表达谱,以及在胚胎期(33、45、65、70和90 d)和出生后阶段(0、9、30、60、120和160 d)骨骼肌中的差异表达进行了比较分析。结果表明,THBS3基因广泛表达于长白猪和通城猪各个组织器官,除胃和肠中的表达存在差异外,其在两个猪种中的组织表达谱基本一致,在肺脏中表达量最高。THBS3基因在长白猪和通城猪胚胎期骨骼肌中的表达水平均显著高于出生后阶段(P<0.05),但胚胎期骨骼肌中的表达模式在两个猪种间存在一定差异,THBS3基因表达峰值出现在长白猪胚胎期45 d,而其在通城猪中表达峰值出现在胚胎期65 d。结果提示,THBS3基因参与了猪骨骼肌生长发育过程及不同类型猪种骨骼肌生长发育异步性的调控。     

Expression Analysis of THBS3 Gene in Different Tissues and Skeletal Muscles at Different Periods from Landrace and Tongcheng Pigs

To study the expression pattern of THBS3 gene in different tissues and during skeletal muscle development, the THBS3 gene expression in different tissues and skeletal muscles during prenatal periods (33, 45, 65, 70 and 90 d) and postnatal periods (0, 9, 30, 60, 120 and 160 d) from Landrace and Tongcheng pigs were detected by Real-time quantification PCR.The results showed that THBS3 gene widely expressed in all tissues examined, exhibiting similar spatial expression patterns with expression peaks in lung in both pig breeds except in stomach and intestine.Moreover, although THBS3 gene showed a significant higher expression level in gestation than after birth in Landrace and Tongcheng pigs (P<0.05), it exhibited different expression patterns between Landrace and Tongcheng pigs, the expression peak was detected at gestation day 45 in Landrace pig, while was detected at gestation day 65 in Tongcheng pig.The results suggested that THBS3 gene involved in skeletal muscle growth and development in pigs, as well as the regulation of asynchronization of skeletal muscle development in different pig breeds.     

Determination of Feeder Cell‐Based Cellular Niches Supporting the Colonization and Maintenance of Spermatogonial Stem Cells from Prepubertal Domestic Cat Testes

Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long‐term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC‐specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome‐shaped morphology, AP activity and expression of SSC‐specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats.