High glucose-induced bovine retinal microvascular pericytes FOXO1A increased expression of_2665

By Pauline Watkins,2014-10-30 19:16
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High glucose-induced bovine retinal microvascular pericytes FOXO1A increased expression of_2665

    High glucose-induced bovine retinal microvascular pericytes FOXO1A increased expression of

     Abstract Objective: To explore the high glucose in cultured bovine retinal capillary pericytes FOXO1A expression. Methods: Primary cultured pericytes using immunocytochemistry and Western blot used to detect high glucose-induced bovine

    retinal microvascular pericytes FOXO1A protein. Results: The retinal capillary pericytes in the 5mmol / L D-glucose after

    72h culture, FOXO1A immunoreactivity mainly in the nucleus, in

    30mmol / L D-glucose or 5μ mol / L H2O2, after 72h culture,

    FOXO1A a significant increase in immunoreactivity , FOXO1A immunoreactivity mainly in the nucleus and cytoplasm; Western blot found that high glucose group and the hydrogen peroxide

    group of cell culture 48,72 h weeks after FOXO1A protein activity and expression than the control group increased significantly. Conclusion: High glucose-induced retinal

    capillary pericytes FOXO1A expression and activity increased significantly.

     Key words Glucose FOXO1A protein expression in pericytes

     Up-expression of FOXO1A in bovine retinal microvascular pericytes induced by glucose

     Abstract AIM: To study the expression of FOXO1A in bovine retinal microvascular pericytes cultured with the high glucose

    medium. METHODS: Primary bovine retinal microvascular pericytes were cultured. FOXO1A expression was measured in bovine retinal microvascular pericytes treated with low glucose (5 mmol / L) , high glucose (30mmol / L) and H2O2 (5μ

    mol / L) in medium containing 20mL / L fetal bovine serum using immunocytochemistry and Western Blot analysis.RESULTS: Slight FOXO1A immunoreactivity was detected the retinal

    microvascular pericytes treated with low glucose after 72 hours distributed in the nucleus. FOXO1A immunoreactivity was significantly increased in the retinal microvascular pericytes treated with high glucose and H2O2 after 72 hours and distributed in the nucleus and cytoplasm of pericytes. Western Blot showed that FOXO1A protein expression levels increased in

    high glucose and H2O2 groups after 48 hours, 72 hours compared with that in low high group and phosphor-FOXO1A protein

    expression levels reduced after 48 hours, 72 hours compared with that in low high group.

     CONCLUSION: These data show that high glucose induced the over-expression of FOXO1A in the retinal microvascular pericytes.

     * KEYWORDS: pericyte; glucose; FOXO1A; protein expression

     0 Introduction

     Diabetic retinopathy is a long-term severe visual

    impairment in diabetic complications, characteristics of change is the function of retinal microvascular abnormalities. Marked by its early loss of retinal capillary pericytes, and the permanent loss of pericytes is the primary damage of diabetic retinopathy. As the weeks cells in the adult retina can not be copied, the loss of pericytes leading to pathological changes in retinal development, including increased permeability, basement membrane thickening, microaneurysm formation, endothelial cell proliferation and

    new blood vessels, eventually leading to blindness [1]. However, there is the loss of the pericytes detailed mechanism is still unclear. Apoptosis is subject to the regulation of various elements in the cell, Fox (forkhead) protein family is officially released in 2000 named in the evolution of uniform that has a spiral - corner - helix motif, 3α spiral and two

    large wing-shaped ring formed (winged) structurally conserved amino acid sequence of the DNA-binding domain of a

    transcription factor [2], in which FOXO sub-family of

    transcription factors, including FOXO1A, FOXO3, FOXO4, FOXO6 and so on, mainly through transcriptional regulation and signal transduction pathway in the animal's growth and development , cell differentiation, metabolism, apoptosis,

    inflammation and immunity play an important role [3]. FOXO1A therefore to explore the role of the retina in diabetes

    contribute to a better understanding of the pathogenesis of diabetic retinopathy.

     1 Materials and methods

     1.1 Materials DMEM medium, fetal bovine serum (FBS),

    trypsin, EDTA were purchased in the United States Gibco Company; DAB chromogenic agents, collagenase ? were purchased

    in the United States Sigma Corporation; α-smooth muscle actin

    mAb, von Willebrand factor polyclonal antibody, FITC labeled

    goat anti-rabbit IgG, Western Blotting light chemical reagents were purchased in the United States Santa Cruz biotechnology company; FOXO1A purchased in the United Kingdom Abcam, Inc.; immunohistochemical staining kit were purchased in the United

    States Vector Laboratories; Phospho-FOXO1A antibody to buy

    biotech companies in Cell Signaling. Fresh bovine eyes from a local abattoir access, ice transport to the laboratory, 4h dealt. Pruning the eye outside the organization, immersion compound iodine solution 10min, containing 100kU / L streptomycin in PBS Green repeated washing. To the anterior segment and vitreous organization, separating out retinal nerve layer, placed containing 100kU / L streptomycin in DMEM in the green, Shredded to the mushy, over-100μm filter, and

    then flip filter, DMEM wash collection of eluate . 800r/min 4 ? centrifuge 5min. Gettering DMEM, adding three times the volume of 625kU / L ? collagenase solution, 37 ? incubation

    terminated after 10min, over 88μm filter to collect offline-

    eluting single-cell suspension. 800r/min 4 ? supernatant

    after centrifugation 5min. After centrifugation, the bottom of the organization to take to join with 200mL / L FBS in DMEM, 50mL / L CO2 37 ? incubation box culture, 48h after the

    replacement of growth medium. 3 ~ 4d for each child using cell scraping the cells would not be required curettage. Be cell fusion time near 0.25g / L trypsin digestion fluid passage to 1:2 in a new culture bottle inoculation. After a passage to take the first-generation cells were 5 × 105 cells / hole in

    the pre-inoculated useful poly-L-L-lysine-coated cover slip in

    24-well plate containing 200mL / L FBS in DMEM culture nearly 80 % of, 40mL / L paraformaldehyde fixed cells, 30min, α-

    smooth muscle actin antibody assay, and von Willebrand factor

    antibody immunocytochemical identification of pericytes.

     1.2 Methods

     1.2.1 Identification of weeks immunofluorescence staining cells in the culture plate pre-poly-L-L-lysine-coated

    coverslip, to be removed when the cell growth near the fusion, 200mL / L paraformaldehyde fixed cells, 30min, each group weeks Cells seeded adequate hydration, 2mL / L TritonX-100

    role in 10min; PBS, after washing, 30mL / L H2O2 solution inhibit endogenous peroxidase, at room temperature for 10min;

    PBS, after washing, 50mL / L and normal serum closed 30min to turn to the serum, dropping a 1:200 rabbit anti-rat anti-α-

    smooth muscle actin (α-SMA) mAb, negative control group plus 0.01mol / L pH value of 7.2 PBS, 4 ? overnight; PBS washing,

    the drop plus two anti-sheep anti-rabbit 1:100 IgG-fluorescein

    isothiocyanate (FITC) were incubated at room temperature away from light 30min; PBS fluorescence microscope after washing, the cells positive for green fluorescence. Rabbit anti-rat von

    Willebrand factor antibody-line immunocytochemistry, methods,

    with the next.

     1.2.2 Chemical analysis of immune cells after passage in order to take the first one-generation cell 5 × 105 cells /

    hole in the pre-inoculated with L-poly-L-lysine-coated

    coverslip in 24-well plate at concentrations of 5.5 mmol / L glucose (control group), 30mmol / L glucose (high glucose group), 5μmol / L H2O2, and 30mmol / L mannitol group (24.5mmol / L 5.5mmol / L glucose) containing 20mL / L FBS in DMEM medium After 72h in culture, 40g / L paraformaldehyde

    fixed cells, 30min, cells seeded in each group weeks to fully hydrated, PBS washing, 2mL / L TritonX-100 role in 10min, PBS

    wash; 30mL / L H2O2 inactivation of endogenous Guo oxide activity 10min after the PBS wash. Normal goat serum closed

    30min, tilting to the excess serum, an anti-drip of 1:200

    rabbit anti-FOXO1A polyclonal antibody, negative control group plus 0.01mol / L pH value of 7.2 in PBS, did not add an anti-,

    4 ? overnight, PBS wash, dropping 1:1 000 biotinylated goat

    anti-rabbit secondary antibody 30min, PBS wash, dropping combination of horseradish peroxidase avidin 30min, PBS, after washing, DAB color. Conventional alcohol dehydration, xylene, after Fengpian transparent.

     1.2.3 Western blot analysis of pass a generation retinal

    capillary pericytes were cultured in 5.5mmol / L (control group), 30mmol / L (high glucose) glucose and 30mmol / L mannitol group (24.5mmol / L 5.5mmol / L glucose) containing 20mL / L FBS in DMEM culture medium 24,48,72 h, in order to

    experiment on the next step. Cells incubated with the provisions of weeks time after the ice after the PBS wash, cell culture bottle by adding an appropriate cell lysate (20mmol / L Tris-HCl, pH 7.5; 150mmol / L NaCl; 10ml / L Triton X-100; 1mmol / L EDTA ; 1mmol / L EGTA; 1mmol / L PMSF; 250mmol / L Na4P2O7; 1mmol / L NaF; 1mmol / L Na3VO4) to collect, 180W, 6min, 0 ? ultrasonic crushing, 5 000r/min,

    centrifugation 5min, take the supernatant, Bradford Determination of protein concentration. The same amount of

    protein in the 25μg, with 2 times the concentration of an equal volume of protein loading buffer, 100 ? C heat 5min, so

    that the full protein denaturation, cooled to room temperature, the protein samples directly on the sample to SDS-PAGE gel-like hole plus . In the upper gel electrophoresis using low-voltage constant pressure, while in the bromophenol blue entered the lower gel electrophoresis using a high-

    voltage constant pressure. Bromophenol blue reached the bottom of the gel can be stopped near the electrophoresis, using

    three Tris - glycine - methanol buffer, the protein

    transferred to nitro-power fiber membrane 1 ~ 1.5h later, with Li Chun Staining of the membrane staining to observe the actual effect of transfer film. Transfer film is completed, in

    the Western washing liquid rinse 2min, exhaustion washing liquid, add containing 50g / L milk powder 0.5g / L Tween-20

    in PBS at room temperature closed liquid shake the bed closed 1h. Exhaustion closed fluid by adding 1:1 000 or 1:1 000 rabbit anti-FOXO1A rabbit anti-Phospho-FOXO1A 1, 4 ?

    overnight incubation, the membrane with Western wash solution placed in the side of the bed slowly shake shake washing 10min. Exhaustion after washing liquid, then add washing liquid, washing 10min. Were washed 3 times. 1:1 000 dropping

    combination of horseradish peroxidase goat anti-rabbit IgG

    secondary antibody at room temperature 2h. Exhaustion after washing liquid, then add washing liquid, washing 10min. Were washed 3 times. Positive bands will be obtained for optical

    density measurement, using an enhanced chemiluminescence kit analysis, optical density area calculation FOXO1A or Phospho-

    FOXO1A protein content.

     Statistical analysis: All experimental data with that using SPSS 10.31 statistical analysis software, two groups

    were compared using t test number, P

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