Percentage Of BrdU-labeled Cells Research Articles

Small interfering RNA targeting PGC-1α inhibits VEGF expression and tube formation in human retinal vascular endothelial cells.

To determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs). hRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O2) or hypoxic (1%, O2) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O2) or hypoxic (1%, O2) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length. PGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes. Blocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.

The role of IGF1 on the differentiation of prolactin secreting cells in the mouse anterior pituitary

IGF1 knockout (IGF1KO) mice show a reduced number of prolactin (PRL) producing cells (PRL cells); however, the role of IGF1 in PRL cell proliferation and differentiation in immature mice is unclear. In this study, ontogenic changes in the percentages of PRL cells, GH producing cells (GH cells), and 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in the anterior pituitary of male IGF1KO mice during the postnatal period were investigated. The percentage of PRL cells in IGF1KO mice was significantly lower at day 20 compared with that in wild-type (WT) mice, while GH cells in IGF1KO mice were significantly increased from day 10. From days 5 to 20, the percentage of BrdU-labeled cells in WT and IGF1KO mice was similar. PRL cells and GH cells are thought to originate from the same progenitor cells, therefore, PRL cells in IGF1KO mice are not able to differentiate because progenitor cells have already committed to be GH cells. However, IGF1, 17beta-estradiol (E(2)), epidermal growth factor (EGF), or IGF1 plus E(2) treatments increased the PRL cell number in the pituitaries in vitro of 10-day-old WT and IGF1KO mice. This fact suggests that these factors are involved in PRL cell proliferation and differentiation. In addition, the increase of PRL cells in IGF1KO mice stimulated by E(2) or EGF was less than that of WT mice. Thus, IGF1 plays a crucial role in PRL cell proliferation and differentiation in mouse pituitaries by regulating the differentiation of progenitor cells and mediating the actions of E(2) and EGF.

Recombinant Human Epidermal Growth Factor Accelerates the Proliferation of Irradiated Human Fibroblasts and Keratinocytes in vitro and in vivo

Irradiation causes the impaired proliferation of cells lining mucosal membranes. Epidermal growth factor (EGF) facilitates proliferation of various skin cells; however, the wound healing effects of EGF on radiation-damaged cells is less well known. To evaluate the effects of recombinant human EGF (rhEGF) on the proliferation of cells following irradiation, we tested two types of fibroblast cell lines and one keratinocyte cell line. The viable cell numbers were significantly increased by rhEGF treatment for 24 h immediately after 8 Gy of irradiation. The most effective dose of rhEGF was 10 nM in all cell lines used in this study. The percentage of BrdU-labeled cells was also significantly increased by rhEGF treatment. To evaluate the effects of rhEGF on radiation-induced oral mucosal damage in BALB/c mice, we systematically injected 1 mg/kg/day EGF for three days after 17 Gy of irradiation. Administered rhEGF ameliorated radiation-induced mucosal damage in vivo. rhEGF significantly increased the epithelial cell layer thickness and the proliferation of basal layer cells as detected by Ki-67 staining. Our results suggest that rhEGF can be a therapeutic treatment for radiation-induced wounds by stimulating the proliferation of fibroblasts and keratinocytes following irradiation.

Effect of mifepristone on proliferation and apoptosis of Ishikawa endometrial adenocarcinoma cells

To determine the mechanism by which mifepristone improves breakthrough bleeding, the effects of mifepristone on proliferation and apoptosis of Ishikawa endometrial carcinoma cells were evaluated in the presence and absence of progestin. Prospective basic research study. Research laboratories for reproductive health at a university medical school. None. Ishikawa cells were cultured in vitro. Mifepristone and/or medroxyprogesterone acetate at various concentrations were added to the cells. Determination of cell proliferation and apoptosis. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine (BrdU) labeling analysis demonstrated that mifepristone inhibited the growth of Ishikawa cells and percentage of BrdU-labeled cells in a time- and dose-dependent manner. Flow cytometry analysis demonstrated that mifepristone at 100 micromol/L, which completely inhibited cell proliferation, increased the proportion of cells in the S phase and diminished the cells in the G2/M phase. Apoptosis was identified by annexin-V-fluorescein isothiocyanate binding and caspase-3 activation. Immunofluorescent double labeling of Ishikawa cells in the absence or presence of mifepristone revealed that BAX protein expression increased and translocated from cytosol to mitochondria. Mifepristone inhibited cell growth by arresting cell cycle progression at S phase, induced apoptosis through caspase-3 activation, and modulated apoptosis regulatory genes BCL2/BAX and FAS/FASLG in Ishikawa cells. Together, these data imply that the improvement in breakthrough bleeding observed with mifepristone might be due to diminished volume of endometrial tissue similar to that seen with endometrial atrophy.

Apoptosis and cell proliferation of small intestinal villi in mitomycin C-treated rats.

Mitomycin C (MMC) therapy often causes toxicity affecting the small intestine. We investigated the relationship between pathological manifestations and cell death, or the proliferation of small intestinal villi in rats treated with MMC. The length of the villi, apoptosis, and cell proliferation were evaluated in the small intestine at 3, 7, and 11 days after MMC treatment by the TUNEL method, BrdU-immunohistochemistry, and transmission electron microscopy. In MMC-treated rats, the body weight decreased until day 7 and recovered from day 8, while most rats had watery stools from days 4 to 7. The villi were the shortest on day 7 and were still shorter on day 11 than in the control group. The highest incidence of TUNEL-positive cells in the small intestinal crypts was observed on day 3, and the number decreased thereafter to reach the control level on day 11. The percentage of BrdU-labeled cells was the highest on day 3 and the lowest on day 7, but recovered to the control level on day 11. The clinical symptoms caused by MMC treatment are consistent with the changes of villous length that reflect the viability of stem cells in the small intestinal crypts about 4 days earlier.

Rapid extension of axons into the CA3 region by adult-generated granule cells

The dentate gyrus continues to produce granule neurons throughout adulthood. The present study examined the extension of axons by adult-generated granule neurons into hippocampal area CA3. We injected the fluorescent retrograde tracers Fast blue (FB) and FluoroRuby (FR) into area CA3 of adult male rats at various times after the administration of 5'-bromo-2'-deoxyuridine (BrdU), a marker of proliferating cells and their progeny. We report that immature granule cells extend axons into CA3 as rapidly as 4-10 days after mitosis. A significant increase in the percentage of BrdU-labeled cells that were labeled with FB or FR was observed by 2 weeks after BrdU administration. This proportion remained roughly constant up to 3 weeks after BrdU-labeling, a time at which markers of a mature neuronal phenotype are expressed. BrdU-labeled cells that contained either FB or FR often were located far from the tracer injection site, indicating that these cells had extended relatively long axons. Collectively these results suggest that adult-generated granule neurons may influence normal hippocampal function, even at a very early stage after their production.

Adult Subventricular Zone Neuronal Precursors Continue to Proliferate and Migrate in the Absence of the Olfactory Bulb

Neurons continue to be born in the subventricular zone (SVZ) of the lateral ventricles of adult mice. These cells migrate as a network of chains through the SVZ and the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into mature neurons. The OB is the only known target for these neuronal precursors. Here, we show that, after elimination of the OB, the SVZ and RMS persist and become dramatically larger. The proportion of dividing [bromodeoxyuridine (BrdU)-labeled] or dying (pyknotic or terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeled) cells in the RMS was not significantly affected at 3 d or 3 weeks after bulbectomy (OBX). However, by 3 months after OBX, the percentage of BrdU-labeled cells in the RMS decreased by half and that of dying cells doubled. Surprisingly, the rostral migration of precursors continued along the RMS after OBX. This was demonstrated by focal microinjections of BrdU and grafts of SVZ cells carrying LacZ under the control of a neuron-specific promoter gene. Results indicate that the OB is not essential for proliferation and the directional migration of SVZ precursors.

Effects of fetal bovine serum, FSH and 17β-estradiol on the culture of bovine preantral follicles

We describe a 7-d culture in droplets of collagen gel of isolated small bovine preantral follicles in medium with or without 10% fetal bovine serum (FBS). In addition, the effect of human recombinant FSH and 17β-estradiol on the morphology and growth of the preantral follicles was investigated in medium without FBS. After culture in medium with 10% FBS, the increase in follicle diameter was 13.1 ± 8.4 μm, the percentage of BrdU-labeled cells was 49.9 ± 11.3 and the number of cells per area granulosa was 11.1 ± 1.8. Omission of serum from the culture medium had no effect on the percentage of labeled cells, but the diameter increase was lower and the cells were smaller. Apparently, serum affects the size of the granulosa cells from small preantral follicles rather than the stimulation of cell proliferation. Addition of human recombinant FSH and/or 17β-estradiol to serum-free medium resulted in a larger diameter increase during culture compared with that of the control. With FSH, this was due to an increase in cell proliferation, while with estradiol this was caused by an increase in granulosa cell size. The effects of simultaneous treatment with FSH and estradiol was simply the combination of their individual effects. In conclusion, small bovine preantral follicles can be cultured for 7 d in the absence of serum and hormones. The follicles increase in diameter and react to FSH with enhanced cell proliferation and to estradiol with an increase in cell size.

Relationship of Invasiveness to Proliferating Activity and to Cytoskeletal Protein Production in Human Neuroepithelial Tumors Maintained in an Organ Culture System: Use of Human Cortex and Dura as Supporting Matrices

Abstract The proliferation and invasiveness of cultured human neuroepithelial tumors were studied. A human malignant astrocytic glioma cell line (U-251 MG) and a medulloblastoma cell line (D283 Med) were maintained for 3 weeks in an organ culture system using adult human brain cortex, dura mater, or Gelfoam sponge as growth matrices. The cells were labeled with bromodeoxyuridine (BrdU) at different time points, and immunohistochemistry was performed for BrdU, glial fibrillary acidic (GFA) protein (in U-251 MG), and neurofilament (NF) protein (in D283 Med). In the U251 MG line, the cells grew successfully in each matrix, forming a fibrillated solid area and a peripheral zone of invasion. The labeling index (LI) expressed as the percentage of BrdU-labeled cells and the percentage of GFA protein-positive cells in the two zones of the explants were analyzed. The LIs in all cultures were significantly higher in the peripheral than in the central zones. On the other hand, the percentage of GFA protein-positive cells in each matrix was greater in the central zone than in the periphery. The LI was inversely correlated with the percentage of GFA protein-positive cells over the areas counted in each growth matrix. GFA protein production in cells grown on cortex and on dura mater was significantly higher than that in cells grown on Gelfoam. In the D283 Med line, the cells formed an aggregated zone, with peripheral cells infiltrating the Gelfoam. This line showed poor growth on human cortex. Cells grown on the dura demonstrated an LI similar to that on Gelfoam, and cells often infiltrated the dura. The LI of these cells was higher in the peripheral than in the central zone of the explants, both on dura and on Gelfoam. The cells showed variable immunopositivity for each NF protein subunit. No apparent difference in the percentage of immunopositive cells for each NF protein subunit was detected between cells grown on dura and on Gelfoam; however, cells grown on dura often demonstrated neurite-like processes. The results indicate that an organ culture system using human brain cortex or dura mater as matrices for in vitro growth is a promising tool for the study of neuroglial differentiation and of the invasiveness of cultured human neuroepithelial tumors.

Relationship of invasiveness to proliferating activity and to cytoskeletal protein production in human neuroepithelial tumors maintained in an organ culture system

The proliferation and invasiveness of cultured human neuroepithelial tumors were studied. A human malignant astrocytic glioma cell line (U-251 MG) and a medulloblastoma cell line (D283 Med) were maintained for 3 weeks in an organ culture system using adult human brain cortex, dura mater, or Gelfoam sponge as growth matrices. The cells were labeled with bromodeoxyuridine (BrdU) at different time points, and immunohistochemistry was performed for BrdU, glial fibrillary acidic (GFA) protein (in U-251 MG), and neurofilament (NF) protein (in D283 Med). In the U-251 MG line, the cells grew successfully in each matrix, forming a fibrillated solid area and a peripheral zone of invasion. The labeling index (LI) expressed as the percentage of BrdU-labeled cells and the percentage of GFA protein-positive cells in the two zones of the explants were analyzed. The LIs in all cultures were significantly higher in the peripheral than in the central zones. On the other hand, the percentage of GFA protein-positive cells in each matrix was greater in the central zone than in the periphery. The LI was inversely correlated with the percentage of GFA protein-positive cells over the areas counted in each growth matrix. GFA protein production in cells grown on cortex and on dura mater was significantly higher than that in cells grown on Gelfoam. In the D283 Med line, the cells formed an aggregated zone, with peripheral cells infiltrating the Gelfoam. This line showed poor growth on human cortex. Cells grown on the dura demonstrated an LI similar to that on Gelfoam, and cells often infiltrated the dura.(ABSTRACT TRUNCATED AT 250 WORDS)

Bromodeoxyuridine in vivo labeling による膀胱腫瘍のS期細胞の検討

At the time of operation, 10 patients with bladder tumors were given an intravenous infusion of bromodeoxyuridine (BrdU, 250 mg) to label the cells in the DNA synthesis phase (S-phase). The removed tumor specimens were stained immunohistologically using anti-BrdU monoclonal antibody. The percentage of BrdU-labeled cells (S-phase cells) were calculated for each specimen, and expressed as Labeling Index (LI). The average LI of G1, G2 and G3 tumors were 0.9%, 2.3% and 11.9%, respectively. LI was almost parallel to pathological grade. In cases of G2 tumor, LI was varied from 0.2 to 6.2%. The higher LI, the more invasive was the tumor. The patient with the highest LI (12.2%) showed lung metastasis postoperatively and died 6 months after operation. S-phase cells were scattered along the basal layer in G1 papillary tumors, and were observed also near the superficial layer in G2 tumors. In cases of non-papillary tumors, S-phase cells were observed irregularly in various layers. This method will give us useful information on evaluation for malignancy and cell kinetics of bladder tumor.