牛磺酸对人脑神经细胞增殖,分化影响的研究

牛磺酸(taurine,C_2H_7NO_3S)的发现至今已有150多年的历史,但对它在动物体内生理功能的认识,还是在近20年内的研究中积累起来的。牛磺酸是一种结构简单,但在体内具有广泛生理功能的β-氨基酸。与其他参与蛋白质合成的氨基酸不同,通常它不做为蛋     

The differentiation of the nerve and glial cells of neocortical transplants placed into the brain of adult rats

Embryonal neural tissue of 17-day-old rat embryos was transplanted into the brain of adult Wistar rats to test the differentiation of transplants with reference to the normal cerebral cortex development. The control and the experimental rats were decapitated 2, 5, 7, 10, 15, 20, 25, and 35 days after the transplantation. Differentiation of neural tissue was studied using monoclonal antibodies against neurofilaments as well as by counting the proportion of differentiated neurons. The glial differentiation was studied by immunohistochemical method using monoclonal antibodies against acid glial fibrillar protein and vimentin. The differentiation of neural cells of transplants proved to be synchronous with the normal ones while the differentiation of glial cells accelerates.     

Engraftment and differentiation of neocortical progenitor cells transplanted to the embryonic brain in utero

Transplantation of neural progenitors or stem cells is a most useful tool to investigate the relative contribution of cell-autonomous mechanisms and environmental cues in the regulation of cell specification and differentiation during CNS development. To assess the capability of neocortical progenitor cells to integrate into foreign brain regions, here we examined the fate of precursor cells isolated from the dorsal telencephalon of E12 ß-actin-EGFP transgenic mouse embryos after heterotopic/heterochronic transplantation to the E16 rat brain in utero. Our observations show that donor cells were able to penetrate, survive and produce mature cell types into wide regions of the host CNS. Namely, EGFP-positive cells acquired site-specific neuronal identities in many telencephalic regions, including neocortex, hippocampus, olfactory bulb and corpus striatum. In contrast, incorporation into more caudal sites was much less efficient. A fraction of donor cells formed large aggregates that remained segregated from the host milieu. Such aggregates contained mature neurons and glia, including some EGFP-negative elements of host origin, and developed the complex organization of the mature nervous tissue. On the other hand, transplanted cells that engrafted in the parenchyma of extratelencephalic regions predominantly generated glial types. The few neurons failed to acquire obvious site-specific phenotypic traits and did not integrate into the local host architecture. Altogether, our observations indicate that E12 neocortical progenitors are already committed towards regional identities and are unable to modify their phenotypic choices when exposed to heterotopic environmental conditions along different rostro-caudal domains of the embryonic CNS.     

Transplantation of stem/progenitor cells of human brain into the brain of adult rats

We studied the development of stem/progenitor cells of the human brain transplanted in the adult rat brain after reproduction in an in vitro tissue culture. It was preliminarily shown by the immunological methods that the stem cells grown in a medium with growth factors formed neurospheres, which were heterogenous and contained both stem and progenitor cells of the human brain. The cells were implanted in the hippocampus, striatum, or lateral ventricle of the rat brain as a suspension or aggregates (neurospheres) and their behavior and differentiation were studies within 10, 20, and 30 days using the morphological and immunochemical methods. The cultured cells of the human brain continued their development in the rat brain, migrated, and formed neurons and astrocytes. The white mater fibers, lateral ventricle wall, and perivascular spaces served as the main pathways of migration. The neuronal differentiation was shown by staining with antibodies to beta-tubulin III, neurofilaments-70, and calbindin. Some growing nerve cells had long processes with growth cones. At the same time, some transplanted cells retained the undifferentiated state within one month after the implantation, as shown by the vimentin expression.     

Proliferation and differentiation of hematopoietic stem cells in hypokinesia]

Using the method of exogenous cloning in vivo of the hemopoietic stem cells of the bone marrow and spleen in the femur and the spleen of mice it was shown that during hypokinesia the kinetics of the stem cells differed in both organs (the spleen and the bone marrow). Differentiation of transplanted stem cells from different sources was unchanged in the spleen, but stem cells of the bone marrow seeding in the femur changed the character of their differentiation in the direction of increase of the erythopoietic function, whereas stem cells of the spleen failed to alter the direction of differentiation.     

Fate of amnion-derived stem cells transplanted to the fetal rat brain: migration, survival and differentiation

We have recently characterized a stem cell population isolated from the rodent amniotic membrane termed amnion-derived stem cells (ADSCs). In vitro ADSCs differentiate into cell types representing all three embryonic layers, including neural cells. In this study we evaluated the neuroectodermal potential of ADSCs in vivo after in utero transplantation into the developing rat brain. A clonal line of green fluorescent protein-expressing ADSCs were infused into the telencephalic ventricles of the developing embryonic day 15.5 rat brain. At E17.5 donor cells existed primarily as spheres in the ventricles with subsets fused to the ventricular walls, suggesting a mode of entry into the brain parenchyma. By E21.5 green fluorescent protein (GFP) ADSCs migrated to a number of brain regions. Examination at postnatal time points revealed that donor ADSCs expressed vimentin and nestin. Subsets of transplanted ADSCs attained neuronal morphologies, although there was no immunohistochemical evidence of neural or glial differentiation. Some donor cells migrated around blood vessels and differentiated into putative endothelial cells. Donor ADSCs transplanted in utero were present in recipients into adulthood with no evidence of immunological rejection or tumour formation. Long-term survival may suggest utility in the treatment of disorders where differentiation to a neural cell type is not required for clinical benefit.     

Survival and neural differentiation of adult neural stem cells transplanted into the mature inner ear

The cochlear sensory epithelium and spiral ganglion neurons (SGNs) in the adult mammalian inner ear do not regenerate following severe injury. To replace the degenerated SGNs, neural stem cell (NSC) is an attractive alternative for substitution cell therapy. In this study, adult mouse NSCs were transplanted into normal and deafened inner ears of guinea pigs. To more efficiently drive the implanted cells into a neuronal fate, NSCs were also transduced with neurogenin 2 (ngn2) before transplantation. In deafened inner ears and in animals transplanted with ngn2-transduced NSCs, surviving cells expressed the neuronal marker neural class III beta-tubulin. Transplanted cells were found close to the sensory epithelium and adjacent to the SGNs and their peripheral processes. The results illustrate that adult NSCs can survive and differentiate in the injured inner ear. It also demonstrates the feasibility of gene transfer to generate specific progeny for cell replacement therapy in the inner ear.     

Combination of metallic impregnation and autoradiography of brain sections. A method for differentiation of proliferating glial cells in the brain of adult rats and mice.

After labeling with 14C-thymidine, frozen sections or paraffin sections of the brain of adult mice or rats were first stained by metallic impregnation and then coated with chrome alum gelatine and with an emulsion layer of about 10 micron. On the autoradiographs 14C-tracks are readily recognized above labelled astrocytes or oligodendrocytes, and these can be well discriminated, if the sections are processed by the silver carbonate method of Rio-Hortega. In contrast, no labelling is obtained, if the gold chloride sublimate method of Cajal is applied.     

Functional differentiation of human brain progenitor cells

Stem cells and progenitor cells derived from the developing human brain have been shown to differentiate into neurons and astrocytes. However, few studies have examined the functional, physiological properties of these differentiated neurons and astrocytes. In this study we have used immunocytochemistry in combination with electrophysiology to examine protein machinery and functional properties of neurons and astrocytes differentiated from human brain progenitor cells (hBPCs).Our results show that serum induces mainly astrocytic phenotype cells that express GFAP and have physiological properties that are typical of astrocytes. hBPCs differentiated with BDNF and PDGF develop mainly into neurons expressing mature neuronal proteins MAP-2, synaptobrevin II and vesicular glutamate transporter I in the process, plus a small population of GFAP-positive radial cells. Based on electrophysiology of BDNF/PDGF-treated cells two classes of cell were identified. Class I cells have functional neuronal properties, including functional voltage-gated Na(+) and K(+) currents, functional AMPA receptors and the ability to generate action potentials. A smaller subpopulation of cells (Class II cells) expresses GFAP and exhibit functional properties of astrocytes, including linear current-voltage relationship and dye-coupling.     

Neural stem cells in the adult human brain

New neurons are continuously generated in certain regions of the adult brain. Studies in rodents have shown that new neurons are generated from self-renewing multipotent neural stem cells. Here we demonstrate that both the lateral ventricle wall and the hippocampus of the adult human brain harbor self-renewing cells capable of generating neurons, astrocytes, and oligodendrocytes in vitro, i.e., bona fide neural stem cells.     

Proliferation, differentiation and characterization of osteoblasts from human BM mesenchymal cells

BACKGROUND: The objective of this study was to isolate osteoprogenitor cells (OPC) from BM mesenchymal stromal cells (MSC) and test their capacity to proliferate and differentiate into osteoblasts. METHODS: Human MSC were separated on a Percoll gradient and cultured in DMEM supplemented with 15% human serum, and characterized by flow cytometric analyzes for CD34, CD13, CD90, CD105 and CD117. To induce differentiation, cultured cells were exposed to 10(-7) m dexamethasone (dexa) and/or 10(-3) m sodium beta-glycerophosphate (beta-GlyP) and 1,25-dihydroxyvitamin D3 (calcitriol) or 9-cis-retinoic acid (9-RA). RESULTS: alkaline phosphatase (AP) activity was detected in cells irrespective of the dexa and/or beta-GlyP treatment. Antigenic phenotypes of MSC were CD34- (more than 99%) and CD13+ CD90+ CD105+ CD117+ (c. 50%). The treatment induced extracellular calcium deposition and gene and protein expression of osteonectin (ON) and bone sialoprotein (BSP): beta-GlyP induced an increase (c. 2.2-fold) of the ON gene and dexa augmented (c. 2.7-fold) the gene expression of BSP II. Gene expression of BSP I reached a maximum at 3 weeks of combined treatment. Osteocalcin gene expression was induced only after additional treatment with calcitriol or 9-RA. Ultrastructural analysis revealed the secretory phenotype of OPC. DISCUSSION: Under appropriate treatment, MSC can give rise to OPC that have the capacity to differentiate into osteoblasts characterized by the expression of osteogenic markers, osteoblastic properties and stromal BM cells phenotypes. These cells may represent a promising material to be utilized in orthopedic cellular therapy.     

Epithelial differentiation of human adipose tissue-derived adult stem cells

Adult human stem cells are employed in novel treatments and bio-artificial devices. Recent studies have identified an abundant source of stem cells termed adipose-derived adult stem (ADAS)-cells in the subcutaneous adipose tissue. Under appropriate culture conditions ADAS-cells differentiate to various cell types, including chondrocytes, adipocytes, and smooth muscle cells. Aiming at epithelial differentiation this study investigated the effect of all-trans retinoic acid (ATRA) on human ADAS-cells. ATRA-induced cytokeratin 18 expression in ADAS-cells and nearly abolished vimentin expression as shown by Western blot. In immunofluorescence, the formation of keratin fibers in ATRA-treated ADAS-cells could be observed. The percentage of ADAS-cells being able to undergo epithelial differentiation as quantified by FACS-analysis was above 80%. Inhibition of cell growth by ATRA was shown using DAPI- and MTT-assays. ATRA can differentiate ADAS-cells toward the epithelial lineage. This finding, along with a previously described neural differentiation, shows that ADAS-cells have epithelial potential.     

Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas

The nature and even existence of adult pancreatic endocrine stem or progenitor cells is a subject of controversy in the field of beta-cell replacement for diabetes. One place to search for such cells is in the nonendocrine fraction of cells that remain after islet isolation, which consist of a mixture of epithelia and mesenchyme. Culture in G418 resulted in elimination of the mesenchymal cells, leaving a highly purified population of nonendocrine pancreatic epithelial cells (NEPECs). To evaluate their differentiation potential, NEPECs were heritably marked and transplanted under the kidney capsule of immunodeficient mice. When cotransplanted with fetal pancreatic cells, NEPECs were capable of endocrine differentiation. We found no evidence of beta-cell replication or cell fusion that could have explained the appearance of insulin positive cells from a source other than NEPECs. Nonendocrine-to-endocrine differentiation of NEPECs supports the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas.     

Induction of differentiation of human leukemia cells]

Human myeloid leukemia cells do not differentiate into functional end-cells but remain in the proliferation pool. Human cell lines can serve as model for hematopoietic cells arrested at different stages of myeloid differentiation and helps to dissect the cellular and molecular events involved in leukemogenesis. Furthermore, several agents have been identified as inducers of differentiation of leukemia cells. Exciting new clinical observation have shown that patients with APL respond well to the treatment with all-trans retinoic acid. RAR-alpha gene was proved to translocated from chromosome 17 to a locus PML on chromosome 15. This new chimeric gene, PML-RAR alpha is extremely important to understand the leukemogenesis of APL.     

Proliferation and differentiation in the human breast during pregnancy

Using multiple immunofluorescence labelling on human breast tissues obtained and freshly frozen at the 12th, 15th, and 18th weeks of pregnancy, we have shown that markers of mammary functional differentiation, milk proteins (beta-casein and kappa-casein), are synthesised by actively cycling (Ki67 positive) as well as non-cycling (Ki67 negative) cells. These results demonstrate that functional differentiation/maturation does not coincide with loss of proliferative potential in human mammary luminal epithelial cells. In addition, we have examined expression patterns of integrin subunits (alpha1, alpha2, alpha3, alpha6, beta1, and beta4) and extracellular matrix components (laminin, fibronectin, collagen I, and collagen IV), since they have been shown to exert influences on mammary differentiation and morphogenesis in vitro. Compared to human breast tissues obtained from non-pregnant women, a decrease in alpha2 labelling on luminal epithelial cells was observed, particularly in expanding acini that showed abundant Ki67 positivity. The expression patterns of other integrin subunits, however, did not change, indicating that the expression patterns of most integrins existing prior to pregnancy are sufficient to support the morphological and functional development associated with milk protein synthesis.