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 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one at a dose of 180mu;g/L,and the cells were incubated for 12 hr at 37℃.After incubation with the drug,the medium was withdrawn,fresh medium was added and incubation was carried out for an additional 24 hr.Apoptosis was monitored by light microscopy,enzyme linked immunosorbent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 efficacy of vitrectomy for vitreous hemorrhage in patients with polypoidal choroidal vasculopathy (PCV). Methods Fourteen patients (14 eyes) of PCV with vitreous hemorrhage diagnosed by routine ophthalmologic examination, A and/or B mode ultrasound, fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA) were enrolled in this study. The patients included eight males (eight eyes) and six females (six eyes), with the mean age of (58.7plusmn;6.0) years. All patients received vitrectomy with silicone oil and C3F8 gas tamponade. There were eight eyes received photodynamic therapy (PDT) after surgery. The retinal reattachment, visual acuity, pathological lesion degree and complications were comparatively analyzed. Results Among 14 eyes, six eyes (42.9%) recovered, seven eyes (50.0%) improved, and one eye (7.1%) aggravated. Ten eyes achieved retinal reattachment after surgery, while four eyes developed retinal detachment after the first surgery. The retina remained attached in these three eyes after silicon oil tamponade, C3F8 gas tamponade and scleral buckling, respectively; but one eye maintained silicon oil without special treatment. Thirteen eyes (92.9%) achieved retinal reattachment finally. Five eyes of them occurred hyphema one to seven days after surgery, but hyphema was absorbed and intraocular pressure was stable after douche of anterior chamber and pharmacotherapy. The vision improved with more than two lines in one eyes, improved with one to two lines in one eye, unchanged in 10 eyes, and decreased in two eyes. Of eight eyes who underwent PDT, abnormal vessels regressed in five eyes, abnormal vessels remained in three eyes. Conclusions Vitrectomy can remove cloudy refracting media for PCV with vitreous hemorrhage. The combined treatment of vitrectomy and PDT can improve or stabilize visual function,is an effective therapy for the PCV with vitreous hemorrhage.
Objective To observe the therapeutic effect of scleral buckling procedure on old retinal detachment. Methods The clinical data of 42 patients (46 eyes), including 24 males (27 eyes) and 18 females (19 eyes), with old retinal detachment treated by scleral buckling procedure in our department were retrospectively reviewed. The duration of the disease ranged from 1 month to 2 years. All the patients were with rhegmatogenous retinal detachment and combined with mainly predominantly-subretinal proliferative vitreoretinopathy (PVR) (stage C), including stage C1 of PVR in 16 eyes (34.8%), stage C2 in 19 eyes (41.3%), and stage C3 in 11 eyes (23.9%). Scleral buckling was performed on 13 eyes (28.3%) and cerclage combined buckling on 33 eyes (71.7%). Sterile air was injected into 36 eyes (78.3%) during the operation, and C 3F 8 was introvitreal injected into 7 eyes (15.2%) after the operation. Results The follow-up duration was from 6 months to 1 year (mean 7.3 months). Retina was completely reattached in 31 eyes (67.4%), and was alleviated obviously in 12 eyes (26.1%). The subretinal fluid increased after the operation with un-reattached retina and vitrectomy was performed in 2 eyes. One eye underwent vitrectomy due to the development of PVR. After the first operation, the curative ratio of retinal detachment was 67.4%, and effective ratio (cure and alleviation) was 93.5%. The visual acuity improved in 28 eyes (60.9%), kept no change in 11 eyes (23.9%), and decreased in 7 eyes (15.2%). Conclusion Reattachment of retina and improvement of visual acuity can be achieved in some degree in some patients with old retinal detachment who undergo simple scleral buckling procedure without vitrectomy. (Chin J Ocul Fundus Dis, 2006, 22: 35-38)
Objective To investigate the effect of hypericin on the activity of protein kinase C (PKC) in cultured human retinal pigment epithelium (RPE) cells in vitro.Methods RPE cells were cultured in standard medium with 10% serum concentrations containing 0.5 to 5.0 μmol/L hypericin with or without preincubation of phorbol 12-myristate 13-acetate (PMA). The activities of cytosolic PKC (c-PKC) and membranous PKC (m-PKC) were assayed by PKC kit. Results The original activities of c-PKC and m-PKC of RPE cells were (35.34±4.10) pmol·min-1·mg-1and (62.52±8.80) pmol·min-1·mg-1.The activity of c-PKC in RPE cells with PMA preincubation decreased rapidly in 5 minutes, with a subsequent slow decrease after 20 minutes and a decrease to 18% of the activity of c-PKC in RPE cells without PMA preinubation after 60 minutes. While the activity of m-PKC in RPE cells with PMA preincubation increased gradually after 5 minutes and reduced after reached the peak at 40 minutes, and then returned to baseline after 60 minutes, eventually decreased below 30% of the control group. When RPE cells were cultured with PMA for 48 hours, the activities of c-PKC and m-PKC were hardly detectable, while RPE cells were cultured with both PMA and hypericin, hypericin could counteract most of down-regulation by PMA. Conclusion Hypericin may inhibit the translocation of PKC in RPE cells,change the activity of PKC, promote the apoptosis of RPE cells likely,and then prevent proliferative vitreoretinopathy. (Chin J Ocul Fundus Dis，2003，19：55-58)
Purpose To investigate bax expression and induction of apoptosis in normal cultured retinal pigment epithelium (RPE) cells ． Methods Cultured human RPE cells were transfected by PMDNA3-hbax，which incoded the whole bax gene and may be induced by Zn2+ under the MTII promoter， through lepofectin mediated protocol．The tested RPE cells were divided into three groups of A，PMDNA3-hbax transfected ;B，PMDNA3 (nude vector) transfected and C ，normal RPE cells．After transfection， DNA gel electrophoreses were perform ed ，the tested RPE cell cycles were analyzed with flow cytometry (FCM)． Results The gel electrophoretogram showed DNA ladder phenomenon，FCM confirmed the apoptosis of RPE cells PMDNA3-hbaxtransfected ， consisting of significant apoptotic peak sited before the G 1 phase and the apoptotic rate was 36%． Conclusion The foreign bax gene can be effectively conducted in to the RPE cell through lepofectinmediated protocol and induced expression ． The foreign bax overexpression may induce the cultured human RPE cell susceptibi lity to apoptosis． (Chin J Ocul Fundus Dis, 2001,17:132-134)
Purpose To study inhibition effects of retinal pigment epithelial (RPE) cells by hyaluronic acid-stimulating activity(HASA)． Methods The cultured human RPE cells added with a series of HASA was measured with cell counting,tetrazolium(MTT)colorimetric assay and tritium labelled thymidine deoxyribosｅ(3H-TdR)incorporation assay.Flow cytometry(FCM)analysis was used to examine RPE cells cycles. Results HASA at concentrations of 12.5～200 mu;g/ml and within 48 hours inhibited RPE cells proliferation with a dose-dependant and time dependant manners.The maximal inhibition rate of RPE cells by HASF was about 48.0%.FCM revealed that the cells in G1 phase increased 7.2% and cells in S phase decreased 9.7%,compared to controls. Conclusion HASA at a certain dose range and period can inhibit RPE cells proliferation. (Chin J Ocul Fundus Dis,1999,15:72-74)
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)