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:In search of the mechanism for photic retinal injury. METHODS:A visible light damage model was established in the primary cultured healthy,adult human RPE cells by using intense fluorescence light (2 400 Lx). RESULTS:Electron microscopy revealed swelling of the mitochondria and obscurity of nuclear membranous structure in the light damaged cells. The decrease or dissolution of organelle,vacuolization of cytoplasm and myelinic degeneration were found in some severely damaged cells. The level of intracellular SOD was decreased to 41% of that of the controls (P＜0.05). CONCLUSION:The structure of the RPE was damaged by the light radiation and the level of intracellular SOD was decreased. These suggested the light damage might be associated with the production of free radicals and the lipid perioxide reaction in membranous structure of cell. （Chin J Ocul Fundus Dis,1996,12: 174-175 ）
Objective To investigate the effects of transformin growth factor-beta (TGF-beta;) and interferon-gamma(IFN-gamma;)on collagen synthesis in human retinal pigment epithelial cells(RPE)． Methods TGF-beta;(0.01～10 ng/ml),recombinant IFN-gamma;(100～10000 Ｕ/ml)or a combination of two were added to cultures of RPE and collagen synthesis of the cells were measured by3 H-proline incorporation assay,indirect immunofluorescence staining and dot-blot hybridization. Results TGF-beta; at 10 ng/ml increased cell uptake of 3 H-proline to 130．87% of controls．It intensified Type IV,I and Ⅲ collagen fluorescent staining as well as mRNA expression．IFN-gamma; at 10000 Ｕ/ml caused 54．72% inhibition of 3 H-proline uptake by RPE,and decreased TypeⅣ collagen fluorescent staining as well as mRNA expression of Type Ⅳ,I and Ⅲ collagens. Conclusion TGF-beta; and IFN-gamma; stimulated and inhibited collagen synthesis of human RPE,respectively．The combination of two had antagonistic effects．IFN-gamma; can be used for inhibition of collagen synthesis of RPE. (Chin J Ocul Fundus Dis, 1999, 15: 245-248)
Objective To investigate the relationship between exposure intensity and illumination time of blue light and replicative senescence of rat retinal pigment epithelial （RPE） cells.Methods Thirtysix 12-14 weeks Wistar rats were kept in the cage with a bluelight bulb ［(450plusmn;10) nm］, and were randomly divided into four groups (no light，nature light，500 lx light and 1000 lx light illumination), each has nine rats. The rats in each group were further divided into three subgroups according to illumination time (one month，two months or three months). Eyeballs were collected after intraperitoneal injection of 10% chloral hydrate. The right eye of each rat was embedded in paraffin and sectioned for hematoxylineosin (HE) staining, while frozen sections of the left eye were stained for the senescence-associated beta;-galactosidase (SA-beta;-Gal). The data were analyzed by SPSS11.5 statistical software.Results The amounts of SA-beta;-Gal positive RPE cells were significantly different between all groups under the same illumination time 17 (P＝0.000）, and between all subgroups of different illumination time with same exposure intensity （P＜0.01）except for the control group (no light). Conclusion Bluelight can induce replicative senescence in rat RPE cells in an intensity and timedependent manner.
Objective To investigate effects of neural retina on development of the structure of outer blood retinal barrier in embryogenesis. Methods The retinal neural epithelium (RNE) and pigment epithelium (RPE) layers of 150, 120 and 90 embryonic chicken eyes incubated for 7,10, and 14 days were peeled off. RNE was used to prepare the culture medium with different conditions (7drcSF3, 10drcSF3, 14drcSF3). RPE cells of 7- and 14-incubated chicken embryos were cultured on laminin-coated transwell filter. The SF3, 7drcSF3, 10drcSF3 , 14drcSF3 medium were used respectively in the apical chamber and SF2 was used in basolateral chamber. After the formation of monolayer, the transepithelial electrical resistance of the RPE was detected. After the fixation of RPE cells, the condition of the tight junction among the cells was observed by immunohis tochemistry and transmission electron microscopy. Results For the RPE cells of 7-and 14-day incubated embryonic eyes, the difference of TER in various medium of SF3/SF2, 7drcSF3/SF2, 10drcSF3/SF2, 14drcSF3/SF2 was statistically significant (P＜0.01). The polarity of RPE cells was induced and the netlike tight junctional strands was urged in the retina-conditioned medium. Conclusion The neural retina may actively promote the formation of the structure of outer blood retinal barrier. (Chin J Ocul Fundus Dis，2004，20：237-240)
Objective To investigate the modulating effect of transforming growth factor beta;2 (TGFbeta;2) and extracellular matrix (ECM) on the transdifferentiation of human fetal RPE (hfRPE) cells into myofibroblast-like cells , and to determine the mechanism of signal transduction. Methods hfRPE cells were cultured on ECM coated or uncoated petri dish with or witho ut TGFbeta;2 in the medium. The expression of alpha;-smooth muscle actin (alpha;-SMA) were detected by immunocytochemistry examination, flow cytometry and Western blotting via calphostin C, genistein, PD98059, and Wortmannin. Results After cultured on ECM coated petri dish with TGFbeta;2 in the medium,there were obvious morphological changes of hfRPE cells including cellular elongating and appearing of actin microfilaments. The results of flow cytometry and immunocytochemistry examination showed that expression of alpha;-SMA obviously increased after TGFbeta;2 was added in the medium in a dose-dependent manner. Compared with which of hfRPE cells cultured on the uncoated surface of culture plates, the total mean fluore scence intensity (TMFI) of hfRPE cells cultured on FN-coated surface increased (38.01plusmn;1.14)% when the stimulation concentration of TGFbeta;2 was 50ng/ml(Plt;0.05). Western blotting further confirmed the effects. The changes mentioned above could be inhibited mostly by protein kinase C (PKC) and calphostin C (10 nmol/L)(Plt;0.01). Conclusion TGFbeta;2 may induce the transdifferentiation of hfRPE cells into myofibroblast-like cells in a dose dependent manner, which could be intensified by FN. These mediated effects of TGFbeta;2 and ECM may act via the PKC signal transduction pathway. (Chin J Ocul Fundus Dis, 2006, 22: 328-332)
Objective To investigate the bloodretinal barrier（BRB）function of porcine retinal pigment epithelial（RPE）cells cultured in vitro. Methods Primary porcine RPE cells were cultured，and the third generation were inoculated in a microporous filter with the filter membrane of polyvinylpyrrolidone（PVP）-free polycarbonate membrane.After 1，2，3 and 4 weeks of culture，the surface of filter membrane was observed by light microscope，and after 2 weeks of culture，the section of filter membrane was observed by light microscope and transmission electron microscope.Transepithelial electrical resistance（TER）was detected and the permeability was measured with fluorescein sodium（FS）and horseradish peroxidase（HRP）. Results Primary porcine RPE cells were cultured successfully.RPE cells converged1week after inoculation; 2 and 3 weeks after inoculation，the density of RPE cells did not changed obviously; 4 weeks after inoculation，the density of RPE cells decreased.The characteristics of polarized growth of monolayer were found in RPE cells on the surface of filter membrane; 2 weeks after inoculation，the TER of RPE cells was（97.44plusmn;11.36）Omega;/cm2 which maintained till the 3rd week after inocubation.After incubated for 30 minutes，only 0.27% of FS and 0.17% HRP reached the inferior filter membrane，and the permeability rate of SF with low molecular weight was higher than which of HRP with high molecular weight. Conclusions The filter with PVPfree polycarbonate membrane may be used to set up the model of RPE cells with polarized growth of monolayer and investigate the barrier function of RPE cells. (Chin J Ocul Fundus Dis, 2006, 22: 188-191)
Objective To investigate the changes of cellular configuration and polarized characteristic of visual pigment during the development of rod cells of neonatal calf in vitro. Methods Retinal cells of neonatal calf were dissociated and cultivated for 10, 20, 30 and 40 days in vitro were used for immunocytochemical analysis. Retinal rod cells were identified by rho4D2 antibody. The configuration of positive cells and rhodopsin molecular distribution were analysed at different cultivated time. The immuno-reactivity of positive cells was measured by image analysis system. Results The rho4D2 immuno-reactive cells included small round cells without protuberance and the cells with protuberance at the peak. At the 10th day after cultivation, the visual pigment immunoreaction was suffusive in the whole cell membrane and apical process; while at the 30th and 40th day, it gathered at the membrane of apical process or one pole of the cells. The results of quantitative analysis showed that the immunoreactive intensity of positive cells at the 20th day after cultivation was ber than that at the 10th day; while there was no significant difference among the immunoreactive intensity at the 20th, 30th, and 40th day. Conclusions Rod cells at the 30th and 40th day after cultivation have the polarized characterization and visual pigment molecular distribution with high level of expressive ability of protein, which are mature neurons. (Chin J Ocul Fundus Dis, 2005, 21: 394-396)
Objective To examine the expression of proliferating cell nuclear antigen (PCNA) of retinal pigment epithelial (RPE) cells, thus assessing the role of mechanism of contact inhibition playing in the process of experimental retinal detachment and reattachemnt.Methods Retinal detachment was produced in 72 cats by subretinal injection of 0.25% solution of healon through a micropipette three weeks after extracapsular lens extraction and vitrectomy. Some of the detached retinae were reattached 24 hours later. At different time, the cats were killed and eye globes were fixed and embeded in paraffin. Histologic sections were processed for immunohistochemistry examination using an antibody to detect PCNA protein. Labeled RPE cells were identified, and the proliferation was quantified in detached and un-detached retinae of detachment group, and also in reattached retinae of reattachment group. The comparsion of PCNA-labeled RPE cells in different groups were analyzed by ANOVA. Results In detached regions of detachment group, PCNA-expression of RPE cells occured within 24 hours, and reached a maximum after 5-6 days, then gradually declined to barely detectable levels after 20 days. Similar tendency was found in reattached retinae, but the number of PCNA-labeled RPE cells was obviously small. Fewer PCNA-labeled RPE cells were found in regions of un-detached retinae in detachment group. The difference of these three groups was significant.Conclusion Proliferation of RPE cells is induced when they lose contact with neural retina, but inhibited after neural retina reattached to RPE cells. It suggests that the mechanism of contact inhibition plays a role in the proliferative process after retinal detachment and reattachment. (Chin J Ocul Fundus Dis，2003，19：20-23)