PURPOSE:To establish methods for cryopreservation of human retinal pigment epithelial cells (RPEs)and cell culture from thawing of frozen cells. METHODS:Primary cultured RPEs or its first or second passages,added with 10 dimetbylsulfoxide,were kept in --20℃ for 1 to 2 hours,and then further froze to -40~C over night before being placed in liquid nitrogen. The frozen cells were thawed in 60℃ within 2 minutes. Trypan blue staining and immunocytochemical staining with anti-human keratin were performed for cell viability and differentiation. The growth curve was also determined by calculating the total number of cells/well/day. RESULTS:The viable rate from frozen RPEs was 90%. No differences were observed for growth activity between cultures from frozen cells and controls. The cells were positive with anti-human keratin staining. The logarithmic growth phase was during I to 4 days and the doubling time yeas 1.55 days. CONCLUSION: Cryopreservation of RPEs in liquid nitrogen can maintain biological activities of cells with normal growth and features after thaw- ing. This will provide cell lines for in vitro experiments and possibly for cell banks for RPE transplantation for some fundus diseases. （Chin J Ocul Fundus Dis,1997,13:157-159）
Objective To establish a method for primary culture of iris pigment epithelial cells(IPE). MethodsEnzyme-Assisted microdissection was used to isolate and cultivate the IPE cells.An identification was made with microscopic and immunohistochemical observations.Results IPE were successfully sultured and showed on differences with RPE in primary culture and subculture.ConclusionEnzyme-Assisted microdissection is a reliable and quick method for the isolation of IPE.
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）
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 effects of platelet-derived growth factor(PDGF) on the expression of α-smooth muscle actin(α-SMA) of cultured human retinal pigment epithelium cells(RPE). Methods Cultured human RPE cells of the 4-6 th passages were divided into two groups: Delbecco′s modified Eagle′s medium (DMEM) and 2%DMEM (20 g/L foeta calf serum+DMEM). PDGF (0,1,50 ng/ml) was added to medium.The expression of α-SMA was detected and quantitatively analyzed by image process of immunofluorescence.Results PDGF stimulated the expression of α-SMA of human RPE cells.In group of DMEM, The rate of RPE of α-SMA expression was 40%-50% and the intension of fluorescence was 8.08 without PDGF. After stimulated by PDGF(1 ng/ml,50 ng/ml), the rates were 80% and 90% respectively, and the intension of fluorescence were 12.35 and 17.23. In 2%DMEM group, The rates of RPE of α-SMA expression were 85% without PDGF, and 95% ,100% respectively treated with PDGF (1 ng/ml,50 ng/ml). The intension of fluorescence was 14.79 without PDGF, and after stimulated by PDGF, they were 16.28 at 1 ng/ml and 21.36 at 50 ng/ml，which was 2 .7 times ber than that in DMEM group without PDGF. Conclusion PDGF could stimulate RPE cells to express α-SMA. (Chin J Ocul Fundus Dis，2003，19：201-268)
Objective To study the characteristics of choroidal circulation in RP. Methods Using ICGA to obse rve 37 cases of RP and compare with healthy volunteers. Results ① The earliest fluorescein filling time of the choroidal arteries in RP group was (14.38plusmn;3.95) seconds,the choroidal veinous in RP group was (17.27plusmn;5.94) seconds,and there was no obvious difference between RP and control group.②The fluorescein failing time of choroidal vein in RP group was (475.75 plusmn;153.70)seconds.③The area of the bright fluorenscence in posterior fundus in RP group was (41.20plusmn;19.99) mm2,and compared with the control group,there was significant difference (P＜0.0001). ④In the mid to late phase during ICGA，in RP group the veillike hypofluorescence was found in 61 e yes (84.7%),plaque hyperfluorescence in posterior fundus in 21 eyes (29.2%),and leakage of heperfluorescence in 4 eyes(5.6%). Conclusion ①The perfusion pressure of choroidal vessels in RP reveals no c hange.②The blood volume of choroidal vessels becomes decreased in RP.③The choroidal capillaries become atrophic in RP.④Choroidal neovascularization may occur in patients with RP. (Chin J Ocul Fundus Dis, 2001,17:26-29)
Objective To investigate the molecular mechanism of apoptosis in cultured human retinal pigment epithelial (RPE) cells. Methods The growth media of confluency human RPE cells were replaced with a daunoblastinacontaining one at a dose of 180mu;g/L,and the cells were incubated for 12 hr at 37℃.After incubation with the drug,the medium was withdrawn,fresh medium was added and incubation was carried out for an additional 24 hr.Apoptosis was monitored by light microscopy,enzyme linked immunosorbent assay(ELISA)and terminal deoxynucleotidyl transferase mediated biotin-dUTP nick-end labelling(TUNEL)staining.The expression of bax and bcl-2 were evaluated by immuncoytochemical staining with anti-human bcl-2 and bax antibodies. Results After the RPE cells treated with daunoblastina,shrinkage of cytoplasm and nucleus was identified.The ratio of nucleus to cytoplasm was increased.TUNEL staining showed that many cells were positive staining.The amount of apoptotic cells was directly proportional to the drug dose.The integral optical desity values for expression of bax inereased by 22.0%(Plt;0.05), and that of bcl-2 did not change significantly(Pgt;0.05). Conclusions During human RPE cell apoptosis induced by daunoblastina,overexpression of bax or low bcl-2/bax ratio were demonstrated.The results suggest that bax and bcl-2 gene expression could play a role in regulation of RPE cell apoptosis. (Chin J Ocul Fundus Dis, 1999, 15: 153-156)
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 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 observe the characteristics of fundus autofluorescence （AF） distribution at the posterior pole in normal subjects. Methods Seventy-nine normal subjects （156 eyes）were studied. Confocal scanning laser ophthalmoscope (cSLO) HRA2 was used to obtain the AF average image at the posterior pole. The distance was calibrated by Digimizer image analysis system. With umbo as the center, the macula was divided into foveola, fovea, parafovea and perifovea areas which with the radius 175, 750, 1250 and 2750 mu;m respectively. Each area was further divided into inferior, superior, temporal and nasal quadrants by two radial lines angle of 90deg;, except for foveola. The AF intensity in four quadrants of different macular regions and optic disc were measured. The AF intensity in vertical and horizontal direction of umbo was also measured. Then the effects of age, eyes, and gender on AF intensity in four quadrants of different macular regions were analyzed. Results There were statistically significant differences in AF intensity among optic disc and four quadrants of macular regions （F=528.648, P＜0.05）. AF distribution was V-type in vertical direction and M-type in horizontal direction. There were statistically significant differences between age groups in foveola, inferior parafovea, temporal parafovea, inferior perifovea, superior perifovea and temporal perifovea （P＜0.01）. There were no statistically significant differences between the two eyes （P＞0.05）. Between genders group, there were statistically significant differences in foveola, superior fovea, inferior fovea, nasal fovea and temporal perifovea （P＜0.05）; no statistically significant differences in the other quadrants （P＞0.05）. Conclusions The distribution of AF intensity is inhomogeneous in macular regions and four quadrants of each region in normal subjects. AF intensity increases with aging. AF distribution is symmetrical in both eyes. There is probably no correlation between gender and AF intensity distribution.