OBJECTIVE:To investigate the index of the rejection of lJle retinal pigment epithelium(RPE)cells transplantation.
METHOD:Allogenic RPE transplantation on rahbits by transcleral technique, the changes of interleukin-2 (IL-2) activity in peripheral blood and the effect of
immunoinhibitor (methylprednisonlone)were detected.
RESLILTS:In the group of simple transplantation,the IL-2 activity in peripheral blood begin to rise in the first day after operation. The peak value occured in the third day,and is still much higher than that of the control group in the 14th day,whereas in the group treated with immunoinhibitor ,there was no obvious difference in the first day after operatlon,in the third day,the IL-2 activity rises slightly,and returned to normal level in the 7th day.
CONCLUSION: After RPE transplantation, the level of IL-2 activity in peripheral blood might serve as an important index to determining and detecting the rejective response.
（Chin J Ocul Fundus Dis,1996,12: 239-241）
Purpose:To evaluate the function of gap junction-mediated intercellular communication in cultured cells of retinal pigment epithelial(RPE) cells from porcine eyes.
Methods:The cultured RPE cells were previously stained by a fluorescent probe 5, 6-carboxy fluorescein diacetate (CFDA) ,and then photobleach the fluorescent molecule in chosed cells. Using laser scanning confocal
microscope (LSCM)to observe fluorescence recovery rate of the RPE cells which located in different condition. The function of gap junction communication was evaluated according to the fluorescence recovery
rate.
Results:The fluorescence recovered after photobleached and the fluorescent density of cells which touching to them descend. The recovery rate per minnte of the cells which the cell number it adjacent to was 1,2 and 3 respectively was 1. 997plusmn;0. 665, 4. 378plusmn;0. 811 and 8. 736plusmn;2. 084. Conclusion:The cultured porcine RPE cells have the function of gap junction communication,and its function proportion is associate to its adjoining cells number.
（Chin J Ocul Fundus Dis,1996,12: 41-42）
Objective To observe the mutation frequency and the characteristics of rentinitis pigmentosa (RP)1 gene in the Chinese patients with autosomal dominant (AD) RP or sporadic RP (SRP), and to evaluate their potential effects on the pathogenesis of RP. Methods Fifty-five members from 7 Chinese families with ADRP, 30 patients with SRP, and 75 healthy adults were recruited. Polymerase chain reaction (PCR) and direct DNA sequencing were used to detect the sequence mutation in the entire coding region and splice sites of RP1 gene. Univariate analysis and multivariate analysis were used to detect the effect of RP1 gene mutation sites on RP. Results Four coding sequence variants were detected in the codes of 852，872，921 and 939 at the exon 4 of RP1 gene. The R872H alteration, which was found in both ADRP families and patients with SRP, showed positive correlation with RP confirmed by the multivariate logistic regression analysis. The P903L alteration was only found in ADRP families but not in the patients with SRP or the healthy adults. Conclusions The R872H alteration in the RP1 gene is likely to increase the risk of RP, and may be a susceptible gene of RP. Whether the P903L alteration is a diseasecausing factor needs to be further studied.
Objective
To investigate the features of ocular fundus of retinal pigment epithelial detachment (PED) in Chinese patients more than 50.
Methods
The clinical data of 31 continuous patients (34 eyes) with PED diagnosed by ocular fundus photochromy, fundus fluorescein angiography (FFA) and indocyanine green angiography ( ICGA ) from Oct, 2001 to Aug, 2004 were analyzed retrospectively.
Results
In 34 eyes with PED, the results of FFA showed serous PED in 18 (52.9%), hemorrhagic PED in 8 (23.5%), and serosanguineous PED in 8 (23.5%); the results of ICGA revealed PED associated with choroidal neovascularization (CNV) in 12 (35.3%), PED associated with ploypoidal choroidal vasculopathy (PCV) in 17 (50.0%), PED associated with both CNV and PCV in 1 (2.9%), and avascular PED in 4 (11.8%).
Conclusions
PED in Chinese patients more than 50 can be associated with CNV, PCV or other avascular diseases, and PCV is the most common intercurrent choroidal vascular disease.
(Chin J Ocul Fundus Dis, 2006, 22: 224-227)
Objective
To clarify the relationship between inhibition of proliferation and cxpression of Ki-67 in cultured human retinal pigment epithelial(RPE) cells.
Methods
The cultured human RPE cells were treated with daunoblastina at a dose of 180 mu;g/L for 12h.Twenty-four hours later,DNA inhibiting rate was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.The expression of Ki-67 was evaluated by immunocytochemical staining technique and image analysis system.Flow cytometry was used to analyse cell cycle.
Results
DNA inhibiting rate was directly proportional to the dosage of daunoblastina.The proportion of the cells positive staining to Ki-67 in the control and the daunoblastina-treated group were 89.3% and 45.6%(Plt;0. 01),and the integral optical density values for expression of Ki-67 in the two groups were 68.1plusmn;6.2 and 27.3plusmn;5.5(Plt;0.01),respectively.The percen tage of cells in G2 phase of cell cycle increased from 8.9% to 29.5%.
Conclusion
G2 block was induced and poliferation was inhibited by daunoblastina in cultured human RPE cells.There is a relatively good correlation between Ki-67 immunostaining and inhibition of RPE cell proliferation.
(Chin J Ocul Fundus Dis，2000，16：1-70)
Objective To observe the expression of proteins in light-injured retinal pigment epithelial (RPE) cells. Methods ARPE19 cells were exposed to the cool white light at the intensity of (2200plusmn;300) Lx for 6 hours to set up the light injured model. Cellular soluble proteins was extracted and analyzed by means of twodimensional electrophoresis to find out the changes of protein map of lightinjured RPE cells. Results Cellular soluble proteins had (390plusmn;10) spots on the map, in which 11 spots had obvious difference between the light injured group and the normal control group. In the lightinjured cells, the expressio of 8 proteins increased, 1 decreased, and 2 disappeared. Conclusion Twodimensional electrophoresis can find out the difference of expression of proteins in lightinjured and normal RPE cells.
Objective To investigate the ingestion, metabolism and subcellular localization of indocyanine green (ICG) in human retinal epithelial (R PE) cells.Methods RPE cells were incubated with 0.25 mg/ml ICG under the condition of 37oC in the camera. The ICG granule and ultrastructure of RPE cells were observed under the electron microscopy after 1, 4, and 24hour incubation, and the ICG autofluorescence was detected by fluorescence microscopy after the incubation for 1, 2, 4, 8, 12, 24, and 48 hours, respectively. The ab sorbency (A value) of ICG solution was measured at 805 nm with ultraviol et/v isible specrtrometer. The standard curve of concentration of ICG was drawn and the related equation of concentration of ICG and the A value was calculated. After being incubated for 1, 2, 4, 8, 12, 24, 48, and 72 hours, respectively, the A value of supernatant fluid was calculated according to the equation. Aft er incubated with ICG for 24 hours, one sample was observed under electron microscope and fluorescence microscope per week to evaluate the metabolizable period of ICG .Results ICG granules were distributed evenly after entering the RPE cells. After incubated with 0.25 mg/ml ICG for 24 hours, no significant change of the ultrastructure of the RPE cells was found. ICG granules accu mulated in the cells as the time goes by and reached the peak after 24 hours, and then they decreased because of the slowdown of the metabolism. Few ICG was still remained in the cells 1 week later Conclusions RPE cells may take in ICG actively. ICG metabolizable period in RPE cells is long, which may be one of the mechanisms of the toxicity of ICG to the retina in the vitreous operation.(Chin J Ocul Fundus Dis，2004，20：179-181)
Objective
To investigate the effects of cytokines on the expression of syndecan-1 in cultured human retinal pigment epithelial (RPE) cells and the signal transduction pathway.
Methods
Reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of syndecan-1 mRNA and protein in normal RPE cells. The expression of syndecan-1 in RPE cells stimulated by different cytokines was detected and quantitatively analyzed by image process of immunofluorescence. The stimulation included 7 and 35 ng/ml tumor necrosis factor (TNF)-alpha; for 24 hours, 1 and 6 mu;g/ml lipopolysaccharide (LPS) for 11 hours, 7 ng/ml TNF-alpha; for 0 to 24 hours (once per 2 hours, and 13 times in total), and 30% supernatant of monocyte/macrophage strain (THP-1 cells) for 3, 14 and 43 hours. The effect of 30% supernatant of THP-1 cells was assayed after pretreated by PD098059［the specific inhibitor of extracellular signal regulated kinase(ERK) 1/2］for 2 hours. After exposed to 30% supernatant of THP-1 cells for 3 hours and treated by 0.25% trypsin for 5 minutes, RPE cells attaching was evaluated by methyl thiazolyl tetrazolium assay.
Results
In normal human RPE cells, expressions of syndecan-1 mRNA and protein were detected, and b syndecan-1 positive yellowish green fluorescence was found in the cell membrane and cytoplasm while light green fluorescence was in the nucleus. As the concentration and stimulated time of TNF-alpha; or LPS increased, the fluorescence intensity decreased（Plt;0.01）, and after exposed to 30% supernatant of THP-1 cells, weaker fluorescence intensity was detected (Plt;0.001). Pretreatment with 50 mu;mol/L PD098059 for 2 hours partly inhibited the effect of THP-1 cells supernatant. After exposed to 30% supernatant of THP-1 cells for 3 hours, the number of attached cells decreased compared with the controls（Plt;0.05）.
Conclusions
TNF-alpha; and LPS down-regulate the expression of syndecan-1 in cultured human RPE cells. The supernatant of THP-1 cells down-regulates the expression of syndecan-1 and lessens the cells attaching, which is at least mediated by ERK 1/2 pathway.
(Chin J Ocul Fundus Dis, 2006, 22: 113-116)
Objective To compare difference of the cross-sectional pathological imaging and quantitative measurement of central serous chorioretinopathy (CSC) between time- and fourier-domain optical coherence tomography (OCT). Methods Consecutive 26 patients (26 eyes) with unilaterial CSC were subsumed. Bilateral eyes of all the patients underwent time- and fourier-domain OCT. Horizontal and vertical line scanning and radial six-line scanning protocols were used for timedomain OCT examination; horizontal and vertical high resolution five-line scanning and macular cube scanning protocols were used for fourier-domain OCT examination. The characteristics of OCT images, retinal segmentation and the quantitative measurement were compared between these two methods. Results Fourier-domain OCT could yield the three-dimensional images of surface of inner limiting membrane (ILM) and RPE. The band of external limiting membrane (ELM) of normal subjects and CSC patients, and the inner segment and outer segment (IS/OS) of normal subjects could be clearly shown by fourier-domain OCT. However, the band of IS/OS disappeared in 65.4% of the CSC patients. The outer boundary of retina was defined in front of the retinal pigmental epithelia (RPE) by fourier-domain OCT. The foveal thickness of normal subjects and CSC patients was (180.50plusmn;12.69) and (158.41plusmn;34.20) mu;m, respevtively. The height of detachment of neural epithelial layer was (245.84plusmn;154.61) mu;m measured by fourier-domain OCT. The band of IS/OS of normal subjects could be clearly shown by time-domain OCT. However, the band of IS/OS disappeared in 73.4% of the CSC patients, which showed no difference with fourier-domain OCT (Z=-0.108, P=0.914). The outer boundary of retina was defined in front of the IS/OS band by OCT. The foveal thickness of normal subjects was (141.16plusmn;12.75) mu;m, which was thinner than that measured by fourier-domain OCT (t=20.671，P=0.000). The foveal thickness and the height of detachment of neural epithelial layer was (146.40plusmn;36.28) mu;m and (240.32plusmn;156.82) mu;m measured by time-domain OCT, respectively, which showed no significant difference with which measured by fourier-domain OCT (t value was from 0.026 to 1.517, P value was from 0.144 to 0.980). Conclusions Fourier-domain OCT yields better visualization of intraretinal layers and more accurate definition of outer boundary of retina than time-domain OCT. Thus the measurements by fourier-domain OCT were more accurate. Moreover, three-dimensional images of CSC shown by fourier-domain OCT enable the comprehensive observation of pathological morphology and location.
Objective
To investigate the regulative characterisitics of growth factors on proliferation of retinal pigment epithelial (RPE) cells in vitro.
Methods
Primary culture and subculture of RPE cells were establised in vitro Tumor.necrosis factor-alpha;(TNF-alpha;),interleukin-1beta;(IL-1beta;) and basic fibroblast growth factor (bFGF) in different concentrations were added to the RPE cells.3 H-thymidine(3 H-TdR) incorporation and a hemocytometer measured DNA synthesis and cell number separately.
Results
After RPE cells were separately treated with TNF-alpha;,IL-1beta; and bFGF,DNA synthesis was increased by 2.74,2.66 and 1.69 folds and cell number was increased by 54%,22% and 7amp; (Plt;0.05) respectively. When two growth factors were combined (TNF-alpha;+bFGF, IL-1beta;+bFGF),3.14,2.84 and 2.57 folds increased DNA synthesis significantly in each group (Plt;0.05). Compared with value by effect of two growth factors,the combination effect of three growth factors (TNF-alpha;+IL-1beta;+bFGF) was still ber (Plt;0.05).
Conclusion
The synergism of growth factors in their action might be one of the important roles in modulating the proliferation of RPE cells.
(Chin J Ocul Fundus Dis,1998,14:95-97)
