妊娠中期猪胎儿神经干细胞分离培养及分化

从妊娠中期猪胎儿(胎龄60 d)脑组织分离培养神经干细胞并诱导其贴壁分化,采用RT-PCR技术检测干细胞及其分化细胞的表面标志.结果显示,神经干细胞中DCX、Hes1、Oct4、CD-90、Nanog、Sox2和Nestin表达阳性;体外诱导的神经干细胞可以分化为星形胶质细胞(表达GFAP)、少突胶质细胞(表达GalC)和神经元细胞(表达NF、NSE和MAP2).结果表明,从妊娠中期猪胎儿脑组织可以分离神经干细胞,神经干细胞具有自我更新和分化潜能.     

绵羊胎盘组织上VEGF及IGF-1受体的表达

采用免疫组织化学的方法鉴定了VEGF受体Flt-1、Flk-1和IGF-1受体IGF-1R在绵羊产后脱落胎盘组织上的表达。结果表明:这些受体均在胎盘组织的滋养层细胞和合体滋养层细胞上表达,此外,Flt-1和Flk-1还在血管内皮细胞上表达。Flt-1和Flk-1同时在胎盘上表达可能说明了2个受体之间有协调作用,同时3种受体都可能参与胎盘生长和营养物质运送。     

新生Wistar大鼠海马神经细胞的体外培养与鉴定

为探讨原代Wistar大鼠海马神经细胞的体外培养方法,本研究取新生24 h内的Wistar大鼠海马组织,无菌剪碎后采用胰酶消化法分离细胞,用含20%胎牛血清的DMEM/F12培养基培养,逐日在倒置相差显微镜下观察。结果发现,从海马组织中分离出的神经细胞具有增殖能力,细胞对数生长期为2~8 d,最长培养30 d;细胞经免疫荧光鉴定Nestin表达呈阳性;免疫组织化学结果显示,在传代培养细胞的胞体和突起均有NF阳性标记物,GFAP抗体和CD68抗体显色均为阴性。由此可见,分离培养的细胞是具有自我更新增殖和多分化潜能的神经元细胞。     

猪胎儿神经干细胞的分离培养和分化

本研究旨在从猪胎儿脑组织中分离培养神经干细胞,观察神经干细胞生长特性和体外增殖、分化特点.利用神经干细胞培养体系,从胎龄30 d的猪胎儿脑组织中分离培养神经干细胞并诱导神经干细胞贴壁分化,采用RT-PCR技术检测干细胞和分化细胞表面标志或相关基因.结果成功分离培养出神经干细胞,神经干细胞具有分化潜能.神经干细胞中Nestin表达强阳性,β-actin、DCX、Hesl、Oct4、Desmin、CD-90、Nanog和Sox2表达阳性;体外诱导的神经干细胞可以分化为星形胶质细胞(表达GFAP)、少突胶质细胞(表达GalC)和神经元细胞(表达NF、NSE和MAP2).结果提示,从猪胎儿脑组织分离神经干细胞具有可行性和有效性,神经干细胞具有自我更新、增殖和分化潜能.     

山羊妊娠早期胎儿胎盘血管形成及相关基因的表达

旨在探究大足黑山羊妊娠早期胎儿胎盘血管发育、分布和血管生成相关因子表达状况,并对血管生成相关因子表达之间和血管发育进行相关性分析。本研究随机选取15只8月龄、体格相近、身体健康的国家级畜禽遗传资源—大足黑山羊青年母羊,经同一种公羊自然交配后(以末次配种当天记为0 d),经剖腹产手术采集妊娠第20、25和30天的胎儿和胎儿胎盘以及经屠宰采集妊娠第45和60天的胎儿和胎儿胎盘作为研究对象,通过免疫组化法评估胎盘血管发育和分布情况,采用qRT-PCR技术检测主要血管生成相关基因的mRNA表达水平,分析血管生成相关基因之间以及与胎盘血管发育分布的相关性。结果显示,胎儿体重和体长随着妊娠的进行逐渐增加,且血管内皮细胞阳性率处于较高水平(>26%)。山羊胎儿胎盘毛细血管总面积/组织区域面积(CAD)逐渐升高;毛细血管总数/单位组织区域面积(CND)在妊娠25~30 d增加,30~60 d降低,毛细血管总面积/毛细血管根数(APC)呈相反趋势;毛细血管总周长/单位组织区域面积(CSD)无显著变化。妊娠早期胎儿胎盘VEGFA的表达量随着妊娠的进行逐渐升高,且与血管分布存在一定的相关性,其受体FLT1和KDR表达量呈先增高后降低的趋势,分别在30和45 d达到峰值。血管生成相关基因ANGPT1/2及其受体TEK和FGF2及其受体FGFR2的表达均在妊娠30 d时较高。综上表明,在妊娠30 d前,胎儿胎盘主要通过毛细血管形成分支促进血管面积的增加;而在30~60 d,主要通过单位血管的增粗促进血管面积的增加,并且血管生成相关基因的表达在妊娠30 d时较高,这可能和子叶开始形成有关。山羊妊娠早期胎儿胎盘中VEGFA可能通过其受体结合与其他血管生成因子共同作用调控胎儿胎盘血管形成。     

关于牛CD34cDNA的克隆及应用

CD34是白细胞抗原，可在不同类型的细胞包括造血细胞中进行表达，抗人，鼠和犬CD34鼠白的单克隆抗体已用于对淋巴造血干细胞的鉴别，本试验克隆出编码牛CD34的cDNA，并测定其核苷酸顺序，推测其蛋白的氨基酸顺序与人，鼠和犬的CD34蛋白的氨基酸顺序的同源性分别为61.1%,56.0%,和66.1%。并以cDNA作为探针进行Northern杂交，探测CD34RNA在胎牛脑，脾，心和肺中的表达。     

猪胎盘特异基因1(PLAC1)的克隆、组织表达及遗传方式研究

旨在研究猪胎盘特异性基因PLAC1的表达规律,并对其基因结构、生物功能和遗传方式进行初步鉴定。本研究以妊娠26、50和95天的猪胎盘组织为材料,利用RACE-PCR及RT-PCR技术克隆猪PLAC1基因及其不同转录本的全长序列,同时通过原位杂交验证其在胎盘中的表达模式。并检测猪PLAC1基因的序列变异。结果表明,猪PLAC1基因存在3种不同的转录本,3种转录本的CDS区完全相同,长525bp,编码174个氨基酸;组织表达谱和原位杂交结果显示,PLAC1基因特异性表达于猪胎盘绒毛膜上皮细胞中,并在不同妊娠时期差异表达,妊娠50和95天PLAC1mRNA的表达显著高于妊娠26天(P<0.01)。猪群中PLAC1基因的SNP基因分型检测结果表明该基因遵循半合基因的遗传方式。本试验结果为研究猪PLAC1基因在胎盘发育过程中的生物功能奠定了基础。     

兔脂肪间质干细胞的分离培养与鉴定

取兔腹股沟皮下脂肪组织，用Ⅰ型胶原酶消化，分离培养脂肪间质干细胞（MSCs），并用免疫组化和体外诱导分化方法对其表面分子标志和多向分化潜能进行鉴定。结果显示，兔脂肪组织中能够分离培养出脂肪MSCs，该类细胞表达CD29、CD44和CD105，不表达CD34、CD45及HLA—DR表面分子标志，并具有可分化为脂肪细胞、神经细胞和成骨细胞的多向分化潜能，证实兔脂肪组织中存在具有多向分化潜能的MSCs。     

Toxic effects of methylmercury,arsanilic acid and danofloxacin on the differentiation of mouse embryonic stem cells into neural cells

This study was performed to assess the neurotoxic effects of methylmercury, arsanilic acid and danofloxacin by quantification of neural-specific proteins in vitro. Quantitation of the protein markers during 14 days of differentiation indicated that the mouse ESCs were completely differentiated into neural cells by Day 8. The cells were treated with non-cytotoxic concentrations of three chemicals during differentiation. Low levels of exposure to methylmercury decreased the expression of GABA_A-R and Nestin during the differentiating stage, and Nestin during the differentiated stage. In contrast, GFAP, Tuj1, and MAP2 expression was affected only by relatively high doses during both stages. Arsanilic acid affected the levels of GABA_A-R and GFAP during the differentiated stage while the changes of Nestin and Tuj1 were greater during the differentiating stage. For the neural markers (except Nestin) expressed during both stages, danofloxacin affected protein levels at lower concentrations in the differentiated stage than the differentiating stage. Acetylcholinesterase activity was inhibited by relatively low concentrations of methylmercury and arsanilic acid during the differentiating stage while this activity was inhibited only by more than 40 µM of danofloxacin in the differentiated stage. Our results provide useful information about the different toxicities of chemicals and the impact on neural development.     

Characterization of CD34⁺ thymocytes in newborn dogs

Using two-color flow cytometry, we characterized CD34(+) cells in the newborn canine thymus. CD34(+) thymic cells comprised approximately 5% of cells recovered by thymus tissue teasing and both large and small thymocytes have been present in this population, the former being 7-12 times more frequent. All CD34(+) cells expressed the pan-leukocyte antigen CD45. The expression of CD44 profile on the large and small CD34(+) thymocytes differed: almost all large CD34(+) cells were CD44(+), while only 75% of small CD34(+) thymocytes co-expressed the CD44 antigen. We have previously described that CD172α is present on the surface of CD34(+) bone marrow cells in dogs. In the thymus, CD172α was expressed on 5-10% and less than 5% of large and small CD34(+) cells, respectively. Some CD34(+) thymocytes also co-expressed T-lineage-specific markers like CD3, CD4, CD8, TCR1 and TCR2. Their expression increased during the large-to-small thymocyte transition. Based on our findings we suggest that thymocyte progenitors enter their primary differentiation center as large CD34(+), CD44(+), CD45(+) and CD172α(+) cells. T-cell specific markers appear on their surface at early stages of differentiation. As the size of progenitors decreases with terminal primary differentiation, the CD34, CD44, and CD172α surface markers are down-regulated.     

Differential patterns of nestin and glial fibrillary acidic protein expression in mouse hippocampus during postnatal development

Intermediate filaments, including nestin and glial fibrillary acidic protein (GFAP), are important for the brain to accommodate neural activities and changes during development. The present study examined the temporal changes of nestin and GFAP protein levels in the postnatal development of the mouse hippocampus. Mouse hippocampi were sampled on postnatal day (PND) 1, 3, 6, 18, and 48. Western blot analysis showed that nestin expression was high at PND 1 and markedly decreased until PND 18. Conversely, GFAP expression was acutely increased in the early phase of postnatal development. Nestin immunoreactivity was localized mainly in the processes of ramified cells at PND 1, but expression subsequently decreased. In contrast, GFAP was evident mainly in the marginal cells of the hippocampus at PND 1, but immunoreactivity revealed satellite, radial, or ramified shapes of the cells from PND 6-48. This study demonstrates that the opposing pattern of nestin and GFAP expressions in mouse hippocampus during postnatal development occur in the early development stage (PND 1-18), suggesting that the opposing change of nestin and GFAP in early postnatal development is important for neural differentiation and positioning in the mouse hippocampus.     

Isolation and characterization of canine umbilical cord blood-derived mesenchymal stem cells

Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III β tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.     

Characterization of Nucleated Cells From Equine Adipose Tissue and Bone Marrow Aspirate Processed for Point-of-Care Use

The objective of this study was to compare nucleated cell fractions and mesenchymal stromal cells (MSCs) from adipose tissue to bone marrow processed by a point-of-care device that are available for immediate implantation. A paired comparison using adipose and bone marrow from five horses was done. The number of nucleated cells, viability, total adherent cells on day 6 of culture and colony-forming unit fibroblasts (CFU-Fs) were determined. Gene expression for markers of stemness, adipogenic, chondrogenic, osteogenic lineage, and collagen formation was measured in total RNA isolated from adherent adipose and bone marrow cells. Day 6 adherent adipose-derived MSC was frozen briefly, whereas day 6 adherent bone marrow–derived MSC was passaged two additional times to obtain adequate cell numbers for chondrogenic, osteogenic, and adipogenic cell differentiation assays. The total cell count per gram was significantly greater for bone marrow, whereas total adherent cells per gram and the CFU-F per million nucleated cells on day 6 were significantly greater for the adipose. In undifferentiated adherent cells, relative gene expression for CD34, adipogenic, and chondrogenic markers and collagen II was significantly lower in the adipose-derived cells. Conversely, expression of collagen I was significantly higher in the undifferentiated adipose-derived cells. Cell density and total RNA were higher in differentiated adipogenic and osteogenic cultures of adipose cells and in chondrogenic cultures of bone marrow cells. This cell preparation method provides a stromal vascular fraction with a large proportion of multipotent MSCs. There are differences in the cells obtained from the two sources. This method can provide an adequate number of multipotent cells from adipose tissue for immediate implantation.     

Expression of neural markers on bone marrow-derived canine mesenchymal stem cells

OBJECTIVE: To evaluate cell surface markers of bone marrow-derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells. SAMPLE POPULATION: Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs. PROCEDURES: Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (beta III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation. RESULTS: Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed beta III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in beta III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Canine bone marrow-derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.     

Could fetal fluid and membranes be an alternative source for Mesenchymal Stem Cells (MSCs) in the feline species? A preliminary study

Domestic cats are preferred models for normal physiology and several human diseases. In the present study feline fetal fluids and membranes were evaluated as possible sources of MSCs. Samples were recovered from 4 pregnant queens after ovarian-hysterectomy. Gestational sacs were separated from uterine wall; after allantoic and amniotic fluids aspiration and chorion-allantois and amniotic membranes separation, all cell lineages were cultured into 25 cm(2) flasks, in DMEM/TCM199, in a 5% CO(2) incubator at 38.5 °C. At passage 3, chondrogenic, osteogenic and adipogenic differentiation ability were evaluated by culturing cell monolayers in differentiating media for 21 days. Cellular characterization with CD90, CD44, CD105, CD73, CD34, CD14, CD45, was performed by flow cytometry. In all samples, adherent fibroblastoid spindle-shaped cells were observed. Positive von Kossa and Alizarin Red staining confirmed osteogenesis. Alcian blue staining of matrix glycosaminoglycans illustrated chondrogenesis, and positive Oil Red O lipid droplets within cell cytoplasm suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105 and negative for CD34, CD14 and CD45; as unexpected and different from human cells, feline cells resulted negative for CD73. Based on this preliminary results, fetal fluids and membranes could represent an alternative sources for mesenchymal stem cells in feline species.