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Objective:To investigate the difference in the radiation sensitivity of hematopoietic stem and progenitor cells (HSPCs) derived from fetal liver and bone marrow.Methods:HSPCs from fetal liver of 14.5 d embryo or bone marrow of 8 week-old mice were isolated to receive a single dose of 5 or 10 Gy irradiation in vitro using a 60Co irradiator. Twelve hours later, the cell apoptosis, mitochondrial reactive oxygen species (ROS) level, colony formation ability and DNA damage in HSPCs were detected. Freshly isolated HSPCs were injected into lethally irradiated CD45.1 + C57BL/6J mice (4.5 Gy+ 5 Gy with an interval of 30 min) Chimerism rate, lineage constitution, and cell cycle were analyzed 12 weeks after transplantation. Results:Compared with bone marrow HSPCs after irradiation, the percentage of apoptosis in fetal liver HSPCs was significantly higher ( t=16.21, 12.27, P<0.05), the level of ROS was dramatically elevated ( t=68.72, 18.89, P<0.05). At 10 Gy, fetal liver HSPCs could not form colonies at all ( t=12.41, 15.67, 9.46, P<0.05). γ-H2AX immunofluorescence staining showed that the DNA damage of fetal liver HSPCs was more severe after irradiation, and the number of Foci formed was significantly higher than that of bone marrow HSPCs ( t=2.27, 2.03, P< 0.05), which indicated that fetal liver HSPCs were more sensitive to radiation. The chimerism rate of transplanted fetal liver HSPCs was lower than that of bone marrow cells ( t=5.84, P<0.05) with a higher proportion of myeloid lineage, suggesting that fetal liver HSPCs had lower in vivo reconstitution capacity than bone marrow HSPCs and were more prone to myeloid differentiation. The cell cycle of bone marrow HSPCs from transplanted chimeric mice was examined, and the proportion of S-phase was significantly higher in the fetal liver group than that in the bone marrow group ( t=2.89, P<0.05). Mitochondrial stress results showed that fetal liver HSPCs had higher basal respiratory capacity ( t=39.19, P<0.05), proton leakage ( t=6.64, P<0.05), ATP production ( t=9.33, P<0.05), and coupling efficiency ( t=7.10, P<0.05) than bone marrow c-Kit + cells, while respiratory reserve capacity ( t=5.53, P< 0.05) was lower than that of bone marrow c-Kit + cells. Conclusions:HSPCs derived from fetal liver display higher radiosensitivty compared with bone marrow HSPCs, laying the foundation for an in-depth illustration of the effects of radiation on hematopoietic stem cells at different developmental stages.
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Background– Gestational diabetes mellitus (GDM) is defined as intolerance of glucose seen during pregnancy and is associated with fetal and maternal morbidity. The aim of our study was to measure various fetal sonographic parameters such as fetal biometry, fetal liver length (FLL), amniotic fluid deepest pocket (AFDP), placental thickness, inter ventricular (IV) septum thickness, Wharton's jelly area and fetal abdominal fat thickness (FAFT) during 21-24 weeks of gestation and comparison of these parameters between Gestational diabetic and normal pregnant women. Tot Methods: al patients selected in our study were 100 in number, of which 50 had GDM and 50 were normal pregnant women. Fetal standard biometry with additional parameters were measured on transabdominal scan from 21-24 weeks. Fetal sonographic measurements and patients characteristics were measured and compared between two groups. P-value was evaluated along with mean, standard deviation, mean difference and confidence interval Patient characteristics and standard fetal Results: biometric parameters were comparable except for femur length (FL), mean femur length was significantly greater in GDM women compared to normal pregnant women (39.20 ± 0.70 vs. 38.36 ± 1.20, p = 0.001). Mean values in GDM vs. normal pregnent women were, fetal placental thickness in mm ( 42.28 ± 2.09 vs. 33.24 ± 1.70, p = 0.001), amniotic fluid maximum vertical pocket in mm (54.96 ± 1.24 vs. 44.46 ± 1.06, p = 0.001), fetal abdomen fat layer thickness in mm (3.59 ± 0.17 vs. 3.46 ± 0.15, p = 0.001), inter ventricular septum thickness in mm (3.71 ± 0.13 vs. 3.63 ± 0.16, p = 0.001), fetal liver length in mm (36.48± 1.15 vs. 31.86 ± 0.90, p = 0.001), Wharton jelly area in mm2 (115.26 ± 1.96 vs. 109.34 ± 4.81, p = 0.001), Fetal sonographic Conclusion: parameters are significantly increased in GDM women compared to normal pregnant women even before 24 weeks. Measurements of these parameters in routine practice could be used to monitor fetal growth and hence can prevent fatal complications.
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[Abstract] Objective To investigate the effect of wingless-type MMTV integration site family member 5b(Wnt5b) gene overexpression mediated by recombinant adenovirus on the differentiation of mouse embryonic liver stem cells and repair of chronic liver injury in mice. Methods Recombinant adenoviruses expressing Wnt5b and green fluorescent protein (GFP) were applied respectively to infect mouse fetal liver stem cells HP14-19, and induced its differentiation and verified the expression of Wnt5b through Real-time PCR and Western blotting. It also applied indocyanine grean(ICG) uptake experiment and periodic acid-schiff(PAS) staining to detect the differentiation ability of HP14-19 into hepatocyte-like cells. The Real-time PCR was chosen to detect hepatocyte markers albumin (Alb) and cytokeratin 18 (CK18) expression. Forty-eight experimental male BALB/ c mice were randomly divided into control group, model group, stem cell treatment group and Wnt5b modified stem cell treatment group. The carbon tetrachloride(CCl
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Objective:To observe the effect of intra-bone marrow injection (IBMI) of fetal liver mononuclear cell suspension on reconstructing the hematopoietic system in mice. Methods : The fetal liver mononuclear cells from the mice with gestational age of 13.5 days were obtained by Ficoll separation technique, and then the hematopoietic stem cells (HSCs) were obtained by magnetic bead sorting. The fetal liver mononuclear cells were injected into the tibias of 137Cs irradiated mice by IBMI. The recipient mice were observed for reconstitution of the hematopoietic system by routine blood examination, blood smear and bone marrow smear. The donor HSCs in the bone marrow of the recipient mice was subjected to flow cytometry 1 year after transplantation. Results: HSCs accounted for 0.171% in the mouse fetal liver mononuclear cells. One week after transplantation of mouse fetal liver mononuclear cells, blood cells derived from donor cells were detected from peripheral blood; in the third week after transplantation, peripheral blood leukocytes, red blood cells and hemoglobin of recipient mice were restored to the pre-irradiation levels; the percentage of donor-derived blood cells in peripheral blood in the fifth week was stable; the HSCs in the bone marrow of recipient mice were derived from donor cells 1 year after transplantation. Conclusion: The murine fetal liver mononuclear cells can efficiently reconstruct the hematopoietic system of mice by IBMI.
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Objective • To explore the differences in morphology, molecular characteristics and biological functions of different types of mouse fetal liver stromal cells. Methods • E13.5 mouse fetal liver stromal cells were obtained by adherent culture, and different cell types were distinguished by morphology and expression of surface markers, such as CD44, CD29, CD106, CD45 and Sca-1. Then microarray assay was conducted by Mouse Genome 430 2.0 Array and analyzed at P3 or <-2 to identify differential expression genes. Canonical pathway and biological function analyses were performed by using ingenuity pathway analysis (IPA software). Results • Spindle-like (CD45+CD106-CD29+CD44+Sca-1-) and fibroblast-like (CD45-CD106+CD29+CD44+Sca-1-) fetal liver stromal cells were isolated in this study according to the cell morphology. The 1 485 highly-expressed genes in spindle-like cells mainly involved in immune and inflammation-related signaling pathways; while the 3 374 highly-expressed genes in fibroblast-like cells mainly involved in extracellular matrix formation and cellular adhesion. Conclusion • Mouse fetal liver stromal cells have strong heterogeneity in biological characteristics and functions, especially in hematopoietic promoting capability.
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Objective To optimize the method of isolating ,culturing and screening fetal mouse liver stem cells in vitro ,and to identify the potential of bi‐directional differentiation .Methods The fetal liver stem cells of mouse were isolated by the density gra‐dient centrifugation and cell difference adherence method ,the proliferation of stem cells was determined by cell plate cloning tech‐nique and MTT method;stem cells were induced for differentiation by adding DMSO and HGF .Results The isolated stem cells showed adherence within 24 h ,which were orbicular‐ovate ,closely packed ,activated within 1~2 weeks ;the positive rates of CD133 , CD49f and EPCAM were (97 .95 ± 1 .21)% ,(92 .71 ± 3 .49)% and (50 .73 ± 3 .45)% respectively ;AFP and CK19 proteins were expressed;red glycogen granules were seen by PAS after induced differentiation;ALB and HNF‐4αwere expressed .Conclusion Fe‐tal hepatic stem cells are successfully isolated by the density gradient centrifugation combined with difference adherence method ,and the isolated cells have strong stemness and proliferation ability ,as well as the ability of bi‐directional differentiation towards hepato‐cytes and bile duct epithelial cells .
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Fetal liver stem cells are not homogeneous but represented mostly by polyploidy hepatocytes and hepatoblasts and likewise early hematopoietic cells. Both cells lately used in regenerative medicine. However, fact remains unclear that, in which period of gestation preferably produces liver parenchyma stem cells and hematopoietic stem cells. In this case practically there is absence of phenotypic characteristics of liver cells in the dynamics of its development. Phenotype of cells reflects the status of its genome, therefore such information are necessary for further development of cell transplantation. Aim: To create CD34+ phenotypic map of fetal stem cells, isolated from abortive material (fetal liver) obtained by means of MTP in gestation periods from 6 to 20 weeks. Methods: Flow cytometry phenotypic characteristics of hematopoietic fetal stem cells: CD34+-cells. Results: Results show that maximum total amount of fetal liver cells could be isolated in the gestation period 20 weeks, minimum - in the gestation period of 6 to 7 weeks. Maximum absolute quantity of CD34+-cells observed in the period of gestation 20 weeks, minimum - in the period of gestation 6 to 7 weeks. Viability of the cells while using our method of isolation is high and varies from 91.5 to 95.5%. The highest percent of CD34+-cells observed in the gestation period 11 weeks, and lowest - in the period of gestation 6 to 7 weeks. Conclusion: Obtaining results allows making preliminary conclusion, that optimal period for isolation of fetal liver hematopoietic CD34+-cells are 11 weeks and 18 to 20 weeks periods of gestation age.
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Cell therapy has a very promising potential for end-stage liver diseases (ESLD).Fetal liver stem/progenitor cells (FLSPCs) have advantages of safety,high survival and proliferation rates,and a small volume,all which make them ideal for liver disease stem cell therapy.During the early phase of our study,we applied a three-step separation method to enrich FLSPCs and obtained a separation efficiency similar to that of the flow-cell sorting method.Additionally,using a fulminant hepatic failure model in rats,we have demonstrated that FLSPCs can contribute to morphological and functional recovery of the liver.This manuscript will discuss how FLSPCs can be induced to accurately differentiate into hepatocytes and cholangiocytes and how FLSPCs maintain self-renewal.The Notch signaling plays a critical role in regulating the differentiation and self-renewal of many types of stem cells.Our previous findings have shown that the Notch signaling plays an important role in FLSPCs differentiation into hepatocytes.Therefore,the Notch signaling might be involved in the differentiation and self-renewal of FLSPCs.We conducted a study on the regulatory effects and relative molecular mechanisms of the Notch signaling on FLSPCs and found the corresponding interfering target,which might become an index for the clinical application of FLSPCs.
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Ginkgo biloba is considered to be an alternative drug for various indications; unfortunately very few studies are available on its side effects. This present study describes the harmful effects of Ginkgo biloba on developing fetal liver. Two experimental groups of six pregnant female mice each were given Ginkgo biloba at human therapeutic dose (A) and a higher dose (B) throughout the gestation period. A third group (C) was taken as a control and given distilled water only. Fetal livers were examined and the effects of the drug observed. There were signs of congestion and fatty change along with dilatation of sinusoids in a dose dependent manner concluding that Ginkgo biloba affects fetal liver.
La Ginkgo biloba es considerada, en varias indicaciones, como un medicamento alternativo; sin embargo, existen pocos reportes disponibles sobre sus efectos secundarios. Este estudio describe los efectos nocivos de Ginkgo biloba en el desarrollo del hígado fetal. Dos grupos experimentales de 6 ratones hembras preñadas recibieron Ginkgo biloba en la dosis terapéutica humana (A) y una dosis más alta (B) por el período de gestación. Un tercer grupo control (C) recibió agua destilada. Los hígados fetales fueron examinados y observados los efectos de la droga. Hubo signos de congestión y degeneración grasa, junto con la dilatación de sinusoides en función de la dosis. Como conclusión la Ginkgo biloba afecta el hígado fetal.
Subject(s)
Humans , Female , Rats , Fetus , Ginkgo biloba/adverse effects , Liver , Liver/pathology , Plant Preparations/adverse effects , Fetus/pathology , Ginkgo biloba/toxicity , Hepatocytes , Hepatocytes/pathology , Photomicrography , Plant Preparations/toxicityABSTRACT
Objective To study the synergistic effect of NKX6. 1 and PDX1 in inducing differentiation of fetal liver-derived mesenchymal stem cells(FL-MSCs) into the pancreatic B cells and to explore the underlying mechanisms, so as to obtain enough islet-like body for transplantation. Methods Recombinant adenovirus vector harboring both PDX1 and NKX6. 1 genes was constructed, and the vector was used to infect FL-MSCs. Then a series of cytokines were used to induce the differentiation of infected FL-MSCs into pancreatic B cells. The expressions of PDX1, NKX6.1 gene, transcription factors NGN3, NeuroDl/Beta2, MafA as well as C-peptide were examined. Results PDX1 and NKX6. 1 were detected in FL-MSCs cells 24 h after infection; cells began to express NGN3, NeuroDl, and MafA and stably expressed pancreatic B cell related factors including insulin after induction. The expression of these molecules was in a certain order. Conclusion PDX1, NKX6. 1 combined with a series of cytokines can effectively induce FL-MSCs to differentiate into pancreatic islet B cells in vitro, which might be through activation of transcription factors NGN3, NeuroDl, and MafA in turn, inducing FL-MSCs to differentiate towards endocrine precursor cells, B endocrine precursor cells and B cells in turn.
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Objective To explore the possibility that rat bone mesenchymal cells (BMSCs) can differentiate into hepatocytes under the affection of fetal liver filtrate. Methods PAS and green indigo dye were used to detect glycogen and differential level of hepatocytes, respectively. The concentration of ALT, AST, ALP in the culture supernatant were served as markers of hepaocyte function. Results Fourteen days after induced by the fetal liver filtrate, BMSCs changed their shapes into polygon, oval or round. Some of BMSCs were positive for AFP and ALB at 7 days after induction, then the number of positive cells increased, and most of BMSCs expressed AFP and ALB till 21days. The PAS reaction and indocyanine green(ICG) intaking also appeared at 7days. Enzyme in supernatant such as ALT, AST, ALP were fristly detected at 7days and peaked at 14days,then the level declined. Conclusion The fetal rat liver filtrate was able to induce BMSCs into cells with function and characteristics of hepatocytes.
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Objective To observe whether appropriate intermittent exercise at the ischemic threshold can safely promote collateral circulation in an ischemic area of the myocardium through the increased expression of vascular endothelial growth factor(VEGF)and its receptor fetal liver kinase-1(Fik-1). Methods A balloon constrictor was surgically implanted in the first obtuse marginal coronary artery(OM1)of miniature pigs.The subjects were divided into 3 groups:a sham-operation group,a pure ischemia group,and an exercise training group.Subjects in the exercise training group performed individualized treadmill programs 30 min daily,5 d per week,for 8 weeks,including 2 two-minute episodes of exercise-induced ischemia.Two pre-exercise episodes of pure ischemia induced by brief OM1 occlusion were also conducted.Only pure ischemia was induced in the pure ischemia group,and the sham-operation group remained sedentary for the experimental period.Relative myocardial blood flow(RMBF)was measured using microspheres.VEGF and Flk-1 expression levels were measured by Western blotting and real time RT-PCR analyses.Cardiac troponin I(ctnI)levels were determined using an enzyme-linked immunosorbent assay(ELISA). Light and electron microscopy were employed to examine myocardia damage in the ischemic area.Results RMBFs in the exercise training group were significantly higher than those in the pure ischemia and sham-operation groups. RMBFs in the pure ischemia group were significantly higher than those in the sham-operation group.The expression of VEGF and Flk-1 proteins and mRNAs in the exercise training group were significantly higher than those in the pure ischemia and sham-operation groups,and the levels in the pure ischemia group were also significantly higher than those in the sham-operation group.After training,no myocardial damage and no ctnI increase was observed in the pure ischemia group.Microscopy revealed no obvious structural changes. Conclusion Intermittent exercise at the isehemia threshold intension can safely promote coronary collateral formation through upregulation of VEGF and Flk-1 expression in the ischemic myocardial area of a porcine model.
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β-Thalassemia is an inheritance anaemia disease due to the defect in β- globin gene. Gene therapy is considered to be the only method which could cure this disease. Adeno-associated virus type 2 (AAV2) has benn gaining more attention as a vector in human gene therapy for its non pathogenic character and broad host range. Although, the efficacy of recombinant AAV2 (rAAV2) in transducing human hematopoietic stem cells has been investigated by researchers, the results were varied from different laboratory. The view was proposed recently that it may be resulted from helper virus in their packaging system. Respecting this, the packaging system without helper virus was used to produce rAAV2. Human early fetal liver hematopoietic cells not only possess many superior peculiarity compared to hematopoietic cells of bone marrow or cord blood, but also the inherent β-globin gene in the cells is not expressed. Studies on the AAV2 transduction of human fetal liver hematopoietic cells and mediated expression of β- globin gene in vivo were performed and the potential role of AAV2 in β-thalassemia gene therapy was analyzed. The rAAV2 containing a normal human β-globin gene (rAAV2-β-globin) without helper virus contamination were produced. The viral titer, purity and the ability of mediating expression of β-globin gene were detected in vitro. Then, human early fetal liver hematopoietic cells were isolated and were further transducted with the rAAV2-β-globin, followed by transplantation into sublethally irradiated BALB/C nude mice to analyze the β-globin gene expression. The results showed that the high titer and purity of rAAV2-β-globin had the ability of mediating β-globin gene expression in vitro. In 8 recipient BALB/C nude mice, the β-globin gene expression were detected in the 2 mice marrow by RT-PCR. The results suggested that rAAV2 could transduce human fetal liver hematopoietic cells and mediate the β-globin gene expression in BALB/C nude mice, meanwhile the expression level of the gene was still rather low. It is necessary to perform further research on AAV2 biology before applying in β-thalassemia gene therapy.
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Objective To investigate the immunological reaction,serum and liver ?-L-iduronidase(IDUA) activity after human fetal liver cells(FLCs) transplantation therapy on mucopolysaccharidosis(MPS) which attribute to IDUA defect.Methods FLCs were transplanted into IDUA deficiency mice.T cell subsets(CD3,CD4 and CD8) was detected by flow cytometry,while IL-2,TNF-? and IFN-? by ELISA,and serum and liver IDUA activity by fluorospectrophotometer at different stage(before transplantation and 5,10,15 days after transplantation).Results Human FLCs survived in IDUA deficiency mice,bringing elevated serum and liver enzyme activity to receptor.T cell subsets,IL-2,TNF-? and IFN-? was significantly higher 5,10 and 15 days after transplantation than those before transplantation(P
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?-Thalassemia is an inheritance anaemia disease due to the defect in?- globin gene. Gene therapy is considered to be the only method which could cure this disease. Adeno-associated virus type 2 (AAV2) has benn gaining more attention as a vector in human gene therapy for its non pathogenic character and broad host range. Although, the efficacy of recombinant AAV2 (rAAV2) in transducing human hematopoietic stem cells has been investigated by researchers, the results were varied from different laboratory. The view was proposed recently that it may be resulted from helper virus in their packaging system. Respecting this, the packaging system without helper virus was used to produce rAAV2. Human early fetal liver hematopoietic cells not only possess many superior peculiarity compared to hematopoietic cells of bone marrow or cord blood, but also the inherent?-globin gene in the cells is not expressed. Studies on the AAV2 transduction of human fetal liver hematopoietic cells and mediated expression of ?- globin gene in vivo were performed and the potential role of AAV2 in?-thalassemia gene therapy was analyzed. The rAAV2 containing a normal human?-globin gene (rAAV2-?-globin) without helper virus contamination were produced. The viral titer, purity and the ability of mediating expression of?-globin gene were detected in vitro. Then, human early fetal liver hematopoietic cells were isolated and were further transducted with the rAAV2-?-globin, followed by transplantation into sublethally irradiated BALB/C nude mice to analyze the?-globin gene expression. The results showed that the high titer and purity of rAAV2-?-globin had the ability of mediating ?-globin gene expression in vitro. In 8 recipient BALB/C nude mice, the ?-globin gene expression were detected in the 2 mice marrow by RT-PCR. The results suggested that rAAV2 could transduce human fetal liver hematopoietic cells and mediate the ?-globin gene expression in BALB/C nude mice, meanwhile the expression level of the gene was still rather low. It is necessary to perform further research on AAV2 biology before applying in ?-thalassemia gene therapy.
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Objective To isolate and culture meshenchymal stem cells from murine fetal liver.Methods flMSCs from mouse fetuses were isolated by adhering to plastic surface.Growth kinetics was determined by growth curve.Cell cycle and phenotype were analyzed by FACSan flow cytometry.Differentiation of adhering cells was induced and identified.Results Homogenous fibroblast-like cells were predominated in culture.The counting of flMSCs increased 2 fold after 24 hours and 83.76%?2.88% of flMSCs were in G0/G1 phases.flMSCs were CD44,CD29 positive but negative for the markers of hematopoietic cells such as CD45,CD11b.flMSCs were able to differentiate along adipogenic,chondrogenic and osteogenic pathways even after being passaged several times.ConclusionflMSCs can be isolated by their plastic-attachable property and can expand without losing their multiple differentiation potential in vitro.flMSCs may offer an appropriate cell source for stem cell therapy.
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The cyclamate, a sweetner substance derived from N-cyclo-hexyl-sulfamic acid, is largely utilized as a non-caloric artificial edulcorant in foods and beverages as well as in the pharmaceutical industry. The objective of this study was to evaluate fetal hepatic karyometric and stereological alterations in the rat fetal liver resulting from the intraperitoneal administration of sodium cyclamate. The livers of ten rats were evaluated, five treated and five controls chosen at random, in which five rats that received from the 10th to 14th days of pregnancy an intraperitoneal daily injection of sodium cyclamate at 60 mg/Kg of body weight during 5 days. At the 20th day of gestation, the animals were removed and weighed, as were their placentas, on a precision balance; the length of the umbilical cords also were measured. After the laboratory processing, semi-seriated 6mm cuts stained with haematoxyline and eosine were performed. In seven karyometric parameters (major, minor, and medium diameters, volume, area, perimeter, and volume-area ratio), the increase was statistically significant in the treated group when compared with control group. Stereological parameters showed in the treated group a significant increase in the cytoplasmatic and cellular volume, and a significant reduction in the nucleus-cytoplasm ratio as well as in the numerical cellular density. These results showed that the sodium cyclamate in pregnant rats led to retardation of fetal development and hepatic-cellular hypertrophy in the offspring, suggesting toxicity in liver of rat fetuses.
El ciclamato, es una substancia derivada del ácido N-ciclo-hexil-sulfámico, bastante usada como edulcorante no calórica en alimentos y bebidas, así como en la industria farmacéutica. El objetivo del trabajo fue evaluar los efectos del ciclamato de sodio en hígados de fetos de ratas, considerándose las alteraciones cariométricas y estereológicas. Fueron utilizadas 10 ratas adultas (Rattus norvegicus) variedad Wistar, con peso medio de 240 g, siendo 5 el grupo control y 5 tratadas con ciclamato de sodio. Entre el 10 y 14 día de la preñez, 5 ratas recibieron una inyección diaria intraperitoneal de 60mg/Kg/día de ciclamato de sodio durante 5 días. En el 20 día, los animales fueron sacrificados y los fetos fijados en solución de Alfac, incluidos en parafina, cortados a 6 µm y teñidos com H-E. Hubo aumento estadísticamente significativo en siete parámetros cariométricos (diámetros mayor, menor y medio, volumen, área, perímetro y relación área/volumen) en el grupo tratado con ciclamato de sodio comparado con el grupo control. Parámetros estereológicos mostraron aumento significativo en los volúmenes citoplasmático y celular y disminución significativa en la relación núcleo/citoplasma y densidad numérica celular. Los resultados mostraron que el uso del ciclamato de sodio en las ratas preñadas causó retardo en el desarrollo fetal e hipertrofia celular hepática en los fetos, sugerente de toxicidad en el hígado fetal de las ratas.
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BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a potent mediator of vascular permeability. Flk-1, one of the receptors for VEGF, is important in vascular development. Increased expression of VEGF is related with reactive astrogliosis, which stimulates the proliferation of neural progenitor cells. VEGF expression increases in the acute phase of cerebral ischemia, however the expression of VEGF together with flk-1 in subacute stage is still unknown. This study is done to demonstrate the spatial/cellular patterns of expression for VEGF/flk-1 up to subacute stages and to find out the role of VEGF in ischemia. METHODS: Transient global ischemia was induced by a 10 min-occlusion/reperfusion of the bilateral carotid arteries in the Mongolian gerbil. Immunohistochemistry and western blot were performed to ensure the expression of VEGF and flk-1 on the day 1, 3, 7, 14, and 28. RESULTS: Both VEGF and flk-1 initially increased at day1, and decreased at day 3. Thereafter, VEGF gradually increased again to the initial level at day 7 and to the peak level after day 14. Flk-1 showed a peak expression at day 14, and then decreased at day 28. Immunohistochemical staining for VEGF showed immunoreactivity mainly on the cytoplasm of neurons and endothelium in cortex and hippocampus at day 1, and neuron, endothelium, and glial cell from day 14 to 28. The distribution and chronological patterns of flk-1 expression were similar to that of VEGF expression. CONCLUSIONS: We suggest that global cerebral ischemia can induce a delayed up-regulation of VEGF and flk-1, which may be associated with neuroangiogenesis and repair process.
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Blotting, Western , Brain Ischemia , Capillary Permeability , Carotid Arteries , Cytoplasm , Endothelium , Gerbillinae , Hippocampus , Immunohistochemistry , Ischemia , Neuroglia , Neurons , Stem Cells , Up-Regulation , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor Receptor-2ABSTRACT
Liver tissuses obtained from 5 human fetuses between 11 weeks and 23 weeks of gestation during the high activity of hepatic hemopoiesis were observed with transmission electron microscope using continuous series of thin sections. The objective of present study was to evaluate ultrastructures of megakaryopoietic cells, the migration of extravascular megakaryocyte into the sinusoidal lumen and the relevence between a migrated megakaryocyte and a Kupffer cell. Immature megakaryocytes were usually observed between growing hepatic laminae and within hepatic sinusoids. A megakaryoblast contained numerous polyribosomes, rather large mitochondria, short tubular elements of rough endoplasmic reticulum and small granules. Moreover, demarcation tubules and a few small specific granules were observed in immature megakaryocytes. The nucleus was mononuclear but frequently indented. With maturation, the nuclei were multilobulated. In the cytoplasm, in contrast to the decrease in polyribosomes and rough endoplasmic reticulum, the numerous specific granules and well -developed demarcation membrane system were predominant. Thereafter cytoplasmic zonation was observed clearly in maturing and mature megakaryocytes. Some megakaryocytes passed through the sinusoidal lining epithelium and into the hepatic sinusoids. The cell to cell interaction was often found as adhesion between migrated megakaryocyte and Kupffer cell, and erythroblasts within megakaryocyte (emperipolesis). These results suggest that intravascular megakaryopoiesis in addition to extravascular megakaryopoiesis occurs to produce platelet during the human fetal liver.
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Humans , Pregnancy , Blood Platelets , Cell Communication , Cytoplasm , Emperipolesis , Endoplasmic Reticulum, Rough , Epithelium , Erythroblasts , Fetus , Liver , Megakaryocyte Progenitor Cells , Megakaryocytes , Membranes , Mitochondria , Polyribosomes , ThrombopoiesisABSTRACT
PURPOSE: This study was undertaken to obtain basic data about the megakaryocyte colony formation of fetal liver cells by using immunocytochemical staining and ex vivo culture with growth factors. METHODS: The mononuclear cells were isolated from fetal liver and bone marrow with idiopathic thrombocytopenic purpura(ITP) and pancytopenia. These mononuclear cells were cultured in MegaCultTM-C(Stem Cell Tech, Canada) media in the presence of growth factors and CFU-Megakaryocyte(CFU-Mk) colonies were counted on day 12. The expansion of CD34+ and CD41+ cell was analyzed by flow cytometry after 5 days incubation using flask culture. RESULTS: The numbers of CFU-Mk colonies of mononuclear cells obtained from fetal liver in the 11th week gestational age were more than those in the 19th week specimens; growth factors could not enhance the colony expansion in all cases. Total numbers of CFU-Mk colony of fetal liver cells were higher than bone marrow from ITP or pancytopenia groups. The numbers of pure or large CFU-Mk colonies of fetal liver cells were also higher than bone marrow specimens. The rate of CD34+ cell expression of fetal liver was increased after flask culture and the enhancement effect of epression was seen only in cases which added thrombopoietin. The rate of CD41+ cell expression of fetal liver was increased after incubation, but the enhancement effect of growth factors was unclear. CONCLUSION: This study revealed good results about the megakaryocyte colony assay of fetal liver mononuclear cells using MegaCultTM-C media. This study suggests that the fetal liver could be a good source of megakaryocytic progenitor cells for clinical application in hematopoietic stem cell transplantation.