High concentration of glucose in cultured human retinal pigment epithelial cells in the cell surface adhesion molecule -1 of
Authors: Han Xiaoxia, Yan-Nian Hui, Song Hu-ping,
WANG Hai-tao, ZHANG Xiao-guang, Liu Bai-jun
【Abstract】 The high concentration of glucose on cultured human retinal pigment epithelial (RPE) cells, intercellular adhesion molecule -1 (ICAM-1) expression. Methods: Using
immunofluorescence assay 5.6mmol / L and 30.0mmol / L glucose effect 1,2,3 d after hRPE cell surface ICAM-1 expression, MTT
was detected by two kinds of concentrations of glucose under the action of 48h hRPE the number of white blood cell adhesion. Results: 30.0 mmol / L glucose 1,2,3 d the role of cell surface fluorescence after hRPE color density of 5.6mmol
/ L glucose under the action of fluorescent coloring 2.4,3.8,4.0 times. 30.0mmol / L glucose after 48h the role of cell-surface adhesion of white blood cell hRPE quantity of 5.6mmol / L glucose group 2.34 times, with statistically
significant difference (P <0.05). Conclusion: High glucose can promote the in vitro hRPE cell ICAM-1 expression, suggesting
that RPE cells under high glucose may be involved in diabetic retinopathy early pathological response.
Key words human retinal pigment epithelial cells
0 Introduction
In recent years, studies have shown that diabetes in the pathogenesis of chronic inflammatory response with a low degree of participation of white blood cells through
intercellular adhesion molecule (ICAM-1) caused by adhesion
and activation in the retina, causing retinal capillary leakage and other inflammatory changes. One direct effect of high concentrations of glucose has also been confirmed by researchers [1]. High glucose can inhibit the proliferation of
microvascular endothelial cells [2], reducing the amount of cell junction protein expression, so that cell shape change, so that large molecules, endothelial cell barrier breakthrough [3]. Retinal pigment epithelial cells as the outer retinal barrier, transfer a large number of retinal sensory layer of glucose to provide energy [4], but also by the blood glucose fluctuations, pathological and functional changes. However, in vitro studies in the RPE cells under the influence of hyperglycemia there is little change in reporting, we have
studied high concentration of glucose on cultured human retinal pigment epithelial ICAM-1 expression, and to explore
whether the RPE cells involved in the early lesions of DR pathological processes.
1 Materials and methods
1.1 The material we do eye cornea transplant donor in primary cultured human RPE (hRPE) cells to establish a limited hRPE cell line, using the first 3 to 6 on behalf of the cells as experimental cell [5], anti-keratin antibodies and
antibodies KG8.13 Immunohistochemical identification of cells. Cell culture using the normal concentration of glucose Dulbecco modified Eagle medium (Dulbecco's modified Eagle's medium, DMEM) powder (Gibco Company, glucose concentration: 5.6mmol / L); bag dry and normal glucose DMEM medium added
24.4mmol D-glucose ( Sigma Corporation), prepared as
containing high concentrations of glucose DMEM culture medium (glucose concentration: 30.0mmol / L).
1.2 Methods
1.2.1 MTT colorimetric rendering the normal concentration
of glucose medium and high concentrations of glucose medium cultured hRPE cell growth curve. Recently was covered with the RPE cells were treated with 2.5g / L trypsin digestion in order to containing 100mL / L newborn calf serum (holly leaf
companies) and normal concentrations of glucose DMEM cell culture medium re-suspension, 5 × 106 / L density were
inoculated into 96 orifice until the adherent cells 24h after
stretching, for use with different concentrations of glucose (? group 5. 6mmol / L, ? group of 30.0mmol / L) DMEM cell
culture medium at 37 ?, 5mL / L CO2 incubator box culture,
replacement of medium every 2d, each equipped with three Vice-
hole. Every 24h randomly selected a 96-well plates, each hole
by adding 5g / L of MTT (Sigma Company) solution, 200μL,
hatching boxes incubated 4h, the supernatant aspirated by adding dimethyl sulfoxide (DMSO) 20μL, shaking the bed in
shock 10min, blank control hole is located on the microplate reader used to read absorbance at 490nm wavelength, according
to the average absorbance value of each picture cell growth curve [6].
1.2.2 Immunofluorescence assay hRPE cell ICAM-1
expression in RPE cells after digestion, to 5 × 106 / L the
density of inoculation 40μL cell suspension was pre-placed in
24-well plate in 8mm × 8mm cover slips, 4h added with 100mL / L fetal calf serum inactivated the normal concentration of glucose DMEM medium 1mL, at 37 ?, 50mL / L CO2 incubator box,
after 24h culture, according to different groups were added with 5.6mmol / L, 30.0mmol / L glucose DMEM culture medium, each including six training holes, each 2d replacement medium. Different groups of cells were removed each day climbing film, 0.01mol / L PBS washed two times, 950mL / L of alcohol -20 ?
fixed 20min, air-drying, -20 ? to save. Fixed cells seeded
returned to room temperature, after 0.01mol / L PBS repeatedly washed, dropping 5mL / L H2O2 / methanol, incubated for 30min at room temperature to inactivate endogenous peroxidase, distilled water rinse after 0.01mol / L PBS wash earthquake ,
dropping 1:50 normal goat serum, at room temperature 30min, tilt go, dropping mouse anti-human ICAM-1 mAb (Santa Cruz
Inc., 1:50), wet box 4 ? overnight, PBS rinsing 3min, 3
times, dropping fluorescein-labeled goat anti-mouse IgG (Santa
Cruz Inc., 1:50), 37 ?, after 30min place, PBS rinsing three times, each time 5min. Buffered glycerol Fengpian fluorescence microscope observation, photography. At the same time replace the first antibody-based PBS blank control and normal goat serum (Zhongshan Company, 1:50) to replace the first alternative to the control antibody.
1.2.3 RPE cell-surface leukocyte adhesion rate of RPE cells, ready to experiment with the former, adjust cell density to 1 × 108 / L, 200μL / hole inoculated in 96-well
plate, 24h post-press group (?, high glucose group ?, and
normal glucose Group ?, sucrose group) to replace 30.0mol / L glucose, 5.6mmol / L glucose DMEM culture solution and 5.6mmol / L glucose 24.4mmol / L sucrose culture medium to cultivate 48h. Set blank control hole and RPE cells in the control holes. Taking the healthy adult peripheral blood, heparin anticoagulation, add equal 30g / L gelatin, 37 ? still
settling 1h, take the upper solution by adding D-Hanks
solution 2 000r/min centrifugation to the supernatant and
repeat wash twice , Hanks solution re-hanging, adjusting the
cell concentration to 1 × 109 / L. By 2 × 105 / conga
leukocyte suspension to 96-well plates, 37 ? incubation
30min, shaking the bed jiggle 1min, gettering is not adherent cells, repeat 1, line MTT colorimetric experiment: the absorbance values of adherent cells, (? A) = absorbance value
of cells in each group (A)-RPE cells, the basis of absorbance (A0), values obtained by statistical analysis, making comparison between the two groups [7].
Statistical analysis: Using SPSS10.0 statistical software, data to x ? s, said, using t test was used for
statistical analysis. P <0.05 statistically significant. The above experiments were repeated 3 times.
2 Results
Newly inoculated primary RPE cells contain more melanin, can not see the nucleus. RPE stretched into an oval shape, polygon, the visible and transparent nucleus. Culture of melanin particles gradually Tuoshi to three generations, melanin particles basically not seen (Figure 1).
2.1 MTT colorimetric results hRPE cells in high glucose group compared with the normal growth curve of cell growth curve of the concentration of glucose was lower, but statistically no significant difference between groups (P>
0.1, Figure 2).
Figure 2 5.6mmol / L, 30.0mmol / L glucose under the action of hRPE cell growth curve
2.2 Immunofluorescence staining in blue-green laser
excitation, the normal concentration of glucose medium group of cell membrane and cytoplasm of the shallow green
fluorescence, high glucose medium group of cytoplasm and
membrane are obvious fluorescence colored fluorescent nuclei Buzhao. Blank control and alternative control group of cells without fluorescent coloring. Application of Image pro-plus
5.0 software to do image analysis and processing: the normal concentration of glucose trace expression of cell fluorescence (0.6146 ? 0.0989), and the 3d was no statistical significance between the fluorescence changes (P> 0.1). High glucose group
cells, strong staining, but increased with time: 1,2,3 d fluorescence intensity were the first 1d normal glucose group 2.4,3.8,4.0 times (Figure 3,4). High glucose group staining intensity of higher than normal concentrations of glucose, and the difference between the two groups was statistically significant (P <0.05 =.
Figure 3 hRPE cells of anti-ICAM-1 fluorescent coloring
A: normal concentration of glucose; B: high-glucose group
Figure 4 different concentrations of glucose medium 2d
hRPE cell fluorescence color × 200
2.3 RPE cell surface adhesion of WBC number of cells high glucose group hRPE cell adhesion of white blood cells absorbance (0.07867 ? 0.00568) higher than normal
concentrations of glucose (0.03367 ? 0.00568) 1.34-fold (P
<0.01), but as osmotic pressure control of 5.6mmol / L glucose 24.4mmol / L sucrose group, the absorbance of white blood cells (0.04005 ? 0.00784) and normal concentrations of
glucose there was no significant difference (P> 0.2).
3 Discussion
As one of the complications of diabetes, diabetic retinopathy clinical diabetes-related blindness is the primary
disease, a serious threat to the eyesight of patients with diabetes [8]. Although the etiology and pathogenesis of diabetes, yet fully clear, but increasing attention in recent years, inflammation theory. The theory of a large amount of clinical data and clinical study indicated that: Diabetes is a natural immunity and low-grade inflammatory disease, diabetic retinopathy was considered to be due to retinal damage caused
by retinal vascular endothelial barrier damage caused by pathological changes. Intercellular adhesion molecule -1
(ICAM-1) occurred in the process of inflammation plays an
important role as a hot Diabetes Study. ICAM-1 is a single-
chain transmembrane glycoprotein, widely distributed, as the leukocyte surface ligand LFA-1 binding, leaving white blood
cells in a variety of endothelial cells and epithelial cell adhesion and involved in inflammatory response [9]. Under normal circumstances cells express little or no expression of ICAM-1, whereas in diabetic patients [10] and diabetic rat model [11], retinal expression of ICAM-1 increase. Reposted
elsewhere in the paper for free download http://
In vitro studies have shown that high blood sugar with a
cytotoxic, Mayumi et al [2] observed that high glucose inhibited the cell growth, PANG Yan et al [12], Wang Changjiang River, etc. [13], Jing Luo et al [14] were observed in high glucose can contribute to vascular endothelial cells
and pericytes apoptosis, while Lee et al [3] further confirmed the role of high glucose under the mouse heart endothelial cell junction protein reduced, and thus cell shape change, can cause leakage of macromolecules. RPE cells, arranged in single layer, to the close connection during a blood - retina outside
the barrier, is involved in proliferative retinopathy in important cell [15]. Inflammatory cytokines, etc., may promote RPE synthesis of vascular endothelial growth factor (VEGF), angiogenesis in the retina, which play a key role in the process. RPE transport a large number of glucose from the choriocapillaris into the retina, and to meet the retinal visual cells in the outer layer of high metabolic activity, so RPE cells more susceptible to the impact of blood glucose
concentration [16]. Grimes et al [17] observed in early diabetic rat RPE cells in the basement membrane surface fold increase in high-glucose-stimulated RPE cells also can make
the changes in glucose transporter protein by protein kinase C
increase RPE cells express VEGF [18]. Experiment, we simply applied a high concentration of glucose as a stimulus and found that high glucose can stimulate the hRPE cell surface ICAM-1 expression increased, and, in high glucose, RPE cell surface adhesion increase in the number of white blood cells. In addition 5.6mmol / L glucose 24.4 mmol / L sucrose solution did not rise to the surface of RPE cell adhesion increase in the number of white blood cells, indicating a high osmotic pressure in itself can not increase the white blood cells in the RPE cell surface adhesion, or high osmotic pressure itself can not promote the RPE cells to produce ICAM-1. We have
observed that in high glucose growth curves under the action
of hRPE glucose concentration under the conditions of normal
cell growth curve, but did not receive statistical support, we believe that in early diabetes, RPE cells may have passed expression of intercellular adhesion molecule - , so that
white blood cells on its surface adhesion, activation, and
thus the release of inflammatory mediators, which in turn activate RPE cells, by releasing VEGF and other factors caused by retinal vascular endothelial cell proliferation, participation and promotion of the proliferative diabetic retinopathy process. In addition, we consider the transport of
glucose due to the role of RPE cells, the ability to withstand high glucose than other cells, strong, simple 30.0mmol / L in high glucose was also not sufficient to affect the RPE cell proliferation, so in early diabetic retinopathy can not detect
pathological RPE cells. Therefore, we speculate that retinal pigment epithelial cells under the effect of high blood sugar, and retinal endothelial cells, together participated in early diabetic retinopathy pathological changes. This gives us a
better understanding of the cause of diabetic retinopathy course provides another line of thought and potential. However, the physiological environment in vivo and in vitro cultivation conditions are not exactly the same, in the body, high blood sugar can act directly on hRPE cells and in vitro caused by the pathological changes consistent with the animal model has yet to be confirmed by further studies.
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