Histopathological findings of cleft palate in rat embryos induced by triamcinolone acetonide

Triamcinolone acetonide (TAC), a synthetic glucocorticoid, induces cleft palate resulting from poor development of palatal shelves in mice. However, TAC has no effect on medial edge epithelial cells (MEE cells) in secondary palatal shelves. In the present study, we examined the relationship between the pathogenesis of cleft palate and the effects on MEE cells and palatal mesenchymal cells in rat embryos/fetuses exposed to TAC. Pregnant Wistar Hannover rats were given TAC intramuscularly at 0.5 mg/kg at gestation days (Day) 12, 13, and 14, then embryos/fetuses were harvested on Days 14.5, 15, 16 and 20. The effects of TAC were as follows; an inhibition of palatal mesenchymal cell proliferation on Day 14.5, a decrease in the density of palatal mesenchymal cells and MEE cells, and expression of epidermal growth factor (EGF) receptors in MEE cells on Day 15, and stratified squamous differentiation of MEE cells with expression of cytokeratin and EGF receptors on Day 16. These findings indicated that TAC inhibited the proliferation of mesenchymal cells and affected the differentiation of MEE cells into stratified squamous epithelia in the palatal shelves of rat embryos. However, these stratified squamous MEE cells partially fused with each other. Thus, we suspected that a major contributing factor to the formation of TAC-induced cleft palate might not be the altered differentiation of MEE cells, but the inhibition of mesenchymal cell proliferation.     

Bone Marrow Mesenchymal Stem Cells: From Characterization of Neural Differentiation to a Possible Therapeutic Use in Neurodegeneration

Bone marrow derived stromal cells are of mesenchymal origin and precursor cells for skeletal tissue components such as chondroblasts and osteoblasts. Furthermore, under experimental conditions, a differentiation potency into myogenic and neuronal cells could be demonstrated. Because of their multipotency these cells represent a population of non-haematogen stem cells that can be regarded as an alternative to human embryonic stem cells for future autologous cell replacement therapies. For a closer look at the differentiation capacity of these cells, rat and human bone marrow stromal cells were isolated from the femur bone and kept in the cell culture applying different cultivation protocols. In a cultivation medium with a serum content of 20%, the majority of these cells express a variety of neuronal markers such as ß-III Tubulin and NeuN as well as the astrocyte marker GFAP, while a minority of about 20% express the marker for neural precursor cells nestin. Cultivation in a chemically defined serum free medium results in the differentiation of a markedly higher percentage of nestin positive neural precursor-like cells. Using bFGF in combination with B27 these cells can be forced to form three dimensionally organized spheres. In order to elucidate a possible therapeutical potency of the bone marrow derived cells the synthesis of neurotrophic factors such as BDNF and NGF were analysed using the ELISA technique. Furthermore, they can be infected using a third generation adenoviral vector with high efficiency and show migratory activity in vitro . After injection of bone marrow derived mesenchymal stem cells into the lateral ventricle of adult rats they adhere to the ependymocytes and pass the ependymal barrier in order to settle in the subventricular space.     

Role of gap junctional intercellular communication (GJIC) through p38 and ERK1/2 pathway in the differentiation of rat neuronal stem cells

Gap junctional intercellular communications (GJIC) contributes to neural function in development and differentiation of CNS. In this study, we have investigated the expression of GJIC during the differentiation of neuronal stem cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neuronal stem cell-derived cells from rat brain. During neuronal stem cell differentiation, expressions of Cx43 and 32 were increased for the duration of 72 hr, however the effect were decreased on the 7d. In the neuronal stem cell-derived cells, pretreatments with p38 MAP kinase inhibitor, SB203580, and MEK inhibitor, PD98059, could protect GJIC against TPA-induced inhibition of GJIC. Our data suggest that GJIC plays an important role during neuronal stem cell differentiation, and ERK1/2 and p38 MAP kinase signaling pathway may be closely related functionally to regulate gap junction in rat neuronal stem cell-derived cells.     

N-acetylserotonin increases cell proliferation and differentiating neuroblasts with tertiary dendrites through upregulation of brain-derived neurotrophic factor in the mouse dentate gyrus

In this study, we investigated the effects of N-acetylserotonin (NAS) on cell proliferation and neuroblast differentiation in the mouse dentate gyrus using anti-Ki67 and anti-doublecortin (DCX) antibodies. Ki67 is expressed in the nucleus or on the surface of chromosomes during all of the active phases of the cell cycle, and DCX is expressed in neuronal precursor cells as well as in immature neurons. At 17 weeks of age, 20 mg/kg of NAS or the same volume of vehicle was intraperitoneally administered once a day for 3 weeks. The animals were sacrificed 2 hr after the last vehicle or NAS treatment. NAS treatment significantly increased the number of Ki67-positive nuclei and DCX-immunoreactive neuroblasts with well-developed dendrites (tertiary dendrites) compared to the vehicle-treated group. However, the number of DCX-immunoreactive neuroblasts without tertiary dendrites was not changed. The administration of NAS also significantly increased the protein levels of brain-derived neurotrophic factor (BDNF) in the dentate gyrus. This result suggests that NAS significantly promotes cell proliferation and the number of differentiating neuroblasts with tertiary dendrites through an increase in BDNF levels in the mouse dentate gyrus.     

Expression of brain-derived neurotrophic factor and tropomyosin-related kinase-B in a bovine jejunal nodular ganglioneuroblastoma

This report describes the morphologic, ultrastructural, and immunophenotypic features of a nodular ganglioneuroblastoma in the jejunum of a 13-month-old Holstein-Friesian heifer. On histologic examination, the mass was composed of clusters of neuroblasts and isolated ganglionic neurons in abundant neurophilic matrix that was surrounded by scanty Schwannian stroma. On ultrastructure examination, the large ganglionic neuron-like cells had unmyelinated neurites. Most ganglionic neuron-like tumor cells expressed neurofilament, neuron-specific enolase, chromogranin A, and S-100, whereas the Schwann-cell-like stromal cells expressed S-100 and vimentin. Both brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase-B (Trk-B) were expressed in ganglionic neuron-like tumor cells, which suggested the activation or reactivation of an embryonic autocrine BDNF/Trk-B pathway that could have prolonged cell survival and promoted differentiation with neurite formation.     

Pathological changes of tracheal mucosa in chickens infected with infectious laryngotracheitis virus

Six-week-old chickens were inoculated via the posterior thoracic air sac with infectious laryngotracheitis virus. Chickens were sacrificed on various days through day 16 postinoculation (PI), and the trachea was examined by scanning electron microscopy (SEM) and light microscopy (LM). The pathological changes observed on day 1 PI were hypertrophy and hyperplasia of goblet cells. From day 3 PI, the epithelial cells protruded collectively and fused to form syncytia, which contained many intranuclear inclusion bodies. Subsequently, epithelial syncytia desquamated, one after another, and connective tissues were exposed in places. Serofibrinous exudate and detritus were abundant on the surface of the exposed connective tissues and seemed to form a pseudomembrane. On day 5 PI, the remaining epithelial cells began to repair the devastated mucosa just under the pseudomembrane. On day 6 PI, microvillus-rich regenerating epithelial cells were arranged like paving stones. On day 8 PI, the epithelial cells proliferated extensively and formed folds with cyst-like structures. By day 16 PI, the tracheal epithelium was covered with cilia and regained its normal histologic appearance.     

The expression of p63 and cytokeratin 5 in mixed tumors of the canine mammary gland provides new insights into the histogenesis of these neoplasms

Cytokeratin 5 and p63 have been described as basal and myoepithelial cell markers in human breast. Mixed tumors of the canine mammary gland have been associated with a myoepithelial origin. Cytokeratin 5 expression has not been evaluated in these tumors. We investigated the relation between cytokeratin 5 and p63 double-immunohistochemical expression in 23 mixed tumors of the canine mammary gland (10 benign mixed tumors and 13 carcinomas arising from benign mixed tumors) and their origin. Cytokeratin 5 and p63 co-expression was observed in myoepithelial cells of benign mixed tumors, as well as in squamous differentiation of carcinoma arising from benign mixed tumors. Though a few interstitial spindle cells of the mesenchymal components expressed both p63 and cytokeratin 5, the basal epithelial cells were labeled only by cytokeratin 5. The co-expression of p63 and cytokeratin 5 in myoepithelial cells and squamous differentiation suggest that, like in human breast, cytokeratin 5 can also be considered a myoepithelial- and squamous-cell differentiating marker in canine tumors. The presence of some interstitial spindle cells stained for p63 and cytokeratin 5 might be associated with a myoepithelial origin of the mesenchymal component of mixed tumors of the canine mammary gland. Moreover, contrary to p63, basal epithelial cells were labeled by cytokeratin 5, indicating that cytokeratin 5 may not represent an exclusive myoepithelial cell marker but also a basal epithelial cell marker in canine mixed tumors. According to these data, basal epithelial cells may be related to the origin of the epithelial component of mixed tumors of the canine mammary gland.     

昆虫神经系统和神经干细胞的研究进展

昆虫的神经系统属于腹神经索型,控制着激素分泌、进食、运动以及支配内脏器官的活动,与脊椎动物的神经系统相比较,其结构简单易于实验操作。对昆虫神经系统的研究,可发现特异性靶标细胞,用于开发新型环保杀虫剂等。此外,昆虫神经干细胞的分裂、分化调控机制与脊椎动物甚至人类有很多的相似性,因而对昆虫神经干细胞的研究可为人类退化性神经疾病研究提供借鉴。本文着重阐述昆虫特别是果蝇的神经系统结构,神经细胞的类型,成神经细胞(neuroblasts,NBs)以及神经干细胞分裂、分化调控机制等方面的研究进展,期望能为开展家蚕神经干细胞的研究提供参考。     

Mouse hepatoblastomas: a histologic, ultrastructural, and immunohistochemical study

Hepatoblastomas from B6C3F1 and BALB/c mice were examined by light and electron microscopy and by immunohistochemical reactions for alpha-fetoprotein, keratin, and vimentin. Tumors occurred in one group of a chronic bioassay for the interaction of diet, genetic strain, and the carcinogen, 2-acetylaminofluorene. Tumors had several populations (including epithelial and mesenchymal cells) in various stages of differentiation. Neoplastic epithelial cells had features of embryonal hepatocytes, such as sparse cytoplasmic organelles, absence of glycogen, abundant free ribosomes, occasional bile canaliculi, and peroxisome-like dense bodies. Embryonal fibroblast-like cells had pleomorphic and folded nuclei with prominent perinuclear chromatin and dispersed cytoplasmic organelles. Fibroblast-like cells were surrounded by bundles of collagen fibrils. Intermediate or transitional types of cells were seen. No tumor cells were immunoreactive for mouse alpha-fetoprotein (AFP) antibody, unlike those in hepatocellular adenomas or carcinomas. Epithelial and mesenchymal tumor cells contained intermediate filaments throughout the cytoplasm; some of these cells stained for keratin but not for vimentin. These findings suggest that mouse hepatoblastomas are derived from bipotential liver blastema cells and are composed of a mixture of several cell populations.     

Isolation,Culture and Multiple Differentiation Induction of Nanyang Bovine Bone Marrow Mesenchymal Stem Cells

The study aimed to build the method of Nanyang bovine bone marrow-derived mesenchymal stem cells (BMSCs) separation culture in vitro, and study its bionomics and the ability of multiple differentiation induction on this basis. Using bone marrow puncture to take rib bone marrow of 3 months calf, isolation and culture BMSCs, subcultured and determined its curve of growth, detected Oct4, Nanog, Sox2 genes expression by RT-PCR technique, then took P3 BMSCs into neural and fat cells to carry out induction differentiation, and took the use of histology staining technique and RT-PCR technique to detect. The results of the BMSCs were uniform sindleshaped in appearance; RT-PCR could dectect the expression of stem cell factor Oct4, Nanog, Sox2. The results showed that an "S" shape growth curve of cell at different generations times, it entered exponential growth stage at the 3rd day generally, and entered plateau after then the 7th day. Into nerve after induction, stained with toluidine blue was apparent throughout the structure of Nissl substance, ENO2 and GFAP genes showed a positive expression. After adipogenic differentiation of ASCs was assessed by oil Red O staining which showed a large number of lipiddroplet, RT-PCR dectected Leptin and PPAR genes showing a positive expression. The tests showed that had successfully isolated from Nanyang bovine BMSCs and it had the potential of multidirectional differentiation.     

南阳牛骨髓间充质干细胞的分离培养及多向分化

本研究旨在建立南阳牛骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)体外分离培养方法,在此基础上研究其生物学特性和多向分化的能力。采用骨髓穿刺法取3月龄小牛的肋骨骨髓,分离培养BMSCs,传代培养并测定其生长曲线,RT-PCR检测Oct4、Nanog、Sox2基因的表达,然后取P3 BMSCs分别向神经和脂肪细胞进行诱导分化,并利用组织学染色技术和RT-PCR技术进行鉴定。结果表明,分离得到的BMSCs大小均匀,多呈梭形的成纤维细胞样生长;RT-PCR可检测到干细胞因子Oct4、Nanog、Sox2的表达;不同代次细胞生长曲线呈S型,一般在第3天时进入指数生长期,第7天后进入平台期;成神经诱导后,甲苯胺蓝染色可见明显的尼氏体结构,RT-PCR检测ENO2和GFAP基因表达呈阳性;成脂肪诱导后,油红O染色后可见大量的脂滴存在,RT-PCR检测Leptin和PPAR基因表达呈阳性。试验证明,成功分离得到了南阳牛BMSCs,且其具有多向诱导分化潜能。     

Malignant fibrous histiocytoma (giant cell type) associated with a malignant mixed tumor in the salivary gland of a dog

A 12-year-old male Boxer dog presented with a 5 x 5 x 7-cm partially encapsulated mass in the right mandibular salivary gland. Histologically, the mass was composed of neoplastic epithelial and mesenchymal cells. The mesenchymal component consisted of two cell populations arranged in different patterns: coalescing nodules of neoplastic mononuclear cells with rare osteoid and numerous osteoclastlike giant cells; and sheets of neoplastic spindle cells intermingled with neoplastic epithelial cells and containing osteoid and well-formed bone trabeculae lined by osteoblasts and few osteoclastlike giant cells. On the basis of these histological features, two malignant salivary tumors were diagnosed: a malignant fibrous histiocytoma (giant cell type) and a malignant mixed tumor. Immunohistochemical studies demonstrated keratin 5 and 8 expression by the neoplastic epithelial cells, indicating a probable salivary ductal origin, and vimentin expression by all mesenchymal elements, suggesting a fibroblastic line of differentiation.     

Cerebral ganglioneuroblastoma in a golden retriever dog

An 8-month-old Golden Retriever dog was euthanatized because of a large cerebral mass extending from the right frontal lobe to the thalamus that was composed of both mature and immature neuronal cells. The better differentiated cells had abundant eosinophilic cytoplasm with prominent Nissl substance and were generally positive for neurofilament and variably positive for synaptophysin. The generally smaller and less-differentiated cells were infrequently positive for proliferating cell nuclear antigen and were negative for any neuronal and glial markers. No apparent glial differentiation of the immature tumor cells was detected. Based on morphologic and immunohistochemical features, the diagnosis of cerebral ganglioneuroblastoma was made. This neoplasm is very rare in all species, especially in the central nervous system, and has never been reported previously in this site in a dog.     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

The Endometrium of Cycling Cows Contains Populations of Putative Mesenchymal Progenitor Cells

Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1–5) and late luteal phases (days 13–18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD‐117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin‐embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c‐KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.     

Appearance of T cell subpopulations in the chicken and embryo retina

Under pathological conditions such as autoimmune encephalomyelitis or autoimmune uveoretinitis, many T cells infiltrate the central nervous system (CNS) and retina. Even in normal condition, a small number of T cells are detected in the CNS. However the characteristics of the T cells are not defined. To investigate the T cell characteristics in a healthy retina, the chicken and the embryo were observed by morphological and immunohistochemical methods. In the chicken retina, T cells were regularly detected, and the main subset was CD-8(+)/ gammadelta cells. Developmentally, CD positive cells appeared on embryonic day 13, and the constituent T cell repertoires became the same as in the chicken by embryonic day 17. Many T cell repertoires were detected on embryonic day 15 and 16. The present results confirm that the retina receives an immunological surveillance by T cells. The composition of T cells in retina is constructed after embryonic day 17. Many ganglion cells die in embryonic days 15 and 16. So the T cell subsets in these periods may involve in autoimmune diseases.     

Expression of MAP kinases and connexins in the differentiation of rat mammary epithelial cells

Gap junctional intercellular communication (GJIC) is involved in the regulation of many cellular processes. MAP kinases are known to affect GJIC and phosphorylation of connexin (Cx). MAP kinases can also be a regulator of cell proliferation and growth. This study was undertaken to show the relevance between expression patterns of Cxs and MAP kinases in rat mammary epithelial cells (RMECs). In order to characterize the RMECs, they were stained with Peanut lectin, which indicates most alveolar epithelial cells, and Thy-1.1 was used as a marker of luminal epithelial cells or myoepithelial cells, respectively. We studied the expression patterns of major gap junction proteins, Cx26, 32, and 43 in RMECs. Western blot analysis demonstrated that Cx26 gradually decreased from day 2, while Cx32 was expressed constantly from day 1 to 14. Cx43 dramatically increased on day 5 and decreased thereafter. The expression patterns and phosphorylation of ERK1/2 and JNK were similar to Cx43, but expression of p38 was like that of Cx32. These results showed that the MAP kinases that comprise ERK1/2, p38, and JNK were involved in regulation of Cxs. Our data suggests that GJIC plays an important role during rat mammary differentiation and that MAP kinases may be closely related functionally to regulate the gap junction.     

崂山奶山羊永生化骨髓间充质干细胞的诱导分化研究

为研究崂山奶山羊永生化骨髓间充质干细胞(BMSCs)的诱导分化潜能,采用P3代BMSCs和P30代TERT-BMSCs进行体外成脂和成神经诱导分化。结果显示,当细胞传至P30代时,TERT-BMSCs生长旺盛;在成脂诱导后,P3代BMSCs和P30代TERT-BMSCs均能观察到细胞中透明脂滴的形成,油红O染料染色后可见脂滴被染成红色;在成神经诱导后,P3代BMSCs和P30代TERT-BMSCs均能形成树突样或三角形的形态,甲苯胺蓝染色后可观察到细胞中尼氏体的存在。综上提示,永生化的崂山奶山羊BMSCs在多次传代后仍具有较强的多向诱导分化能力,这为永生化MSCs的广泛应用提供了理论依据。     

Intercellular junctions in the developing mammalian thymus.

Lymphopoiesis in fetal thymuses of mice and rabbits was studied to try to correlate heterogeneity of lymphocytes with heterogeneity of embryonic origin. The thymuses of fetal mice of 11 to 14 days gestation and of fetal rabbits of 18 and 19 days gestation were examined by electron microscopy. At the time of earliest appearance of lymphoid cells in both species desmosomes were seen on both epithelial and lymphoid cells. These desmosomes persisted for several days, then disappeared from completely differentiated lymphocytes in both species. The similarity between epithelial and lymphoid cells in their earliest developmental stages suggests a common precursor cell for both.     

CRISPR系统促进脂肪干细胞成骨分化与抑制成脂分化的研究

本研究旨在优化组织培养法分离小鼠脂肪间充质干细胞（adipose-derived stem cells,ASCs）,为研究成骨分化和成脂分化在间充质干细胞分化过程中的相互影响奠定基础。通过细胞形态学观察、细胞生长曲线和流式仪器检测所分离获得的间充质干细胞的特性,利用CRISPR-dCas9系统在快速促进间充质干细胞成骨分化的前提下观察其对成脂分化的影响,并通过生化染色、实时荧光定量PCR和免疫细胞学等手段进行分析。结果显示,接种3~5 d后可见细胞从组织块周围爬出,光镜下可见细胞形态多为成纤维细胞样的梭形细胞,且形态单一均匀,具有较高的爬出率,可以大大提高脂肪间充质干细胞的分离效率;通过CRISPR-dCas9系统激活Runx2和Osterix基因后可以促进间充质干细胞的成骨分化,实时荧光定量PCR及油红O染色结果显示,CRISPR-dCas9系统可以同时抑制间充质干细胞的成脂分化;通过CRISPR-dCas9-KRAB系统同时抑制成骨相关基因Runx2和Osterix后可以促进成脂分化。本研究利用组织贴壁法成功获得了高纯度的脂肪间充质干细胞,具有间充质干细胞的特性和分化能力;利用CRISPR系统可以同时过表达Runx2和Osterix两个基因,可以在进成骨分化的同时抑制成脂分化,表明成脂分化和成骨分化的相关性,为基因编辑在间充质干细胞诱导分化和临床应用方面提供了新的思路和方法。