Objective To observe the effect of epithelial-mesenchymal transdifferentiation (EMT) of human retinal pigment epithelial (RPE) cells induced by vitreous humor in vitro. Methods The third to fifth passage cultured RPE cells were divided into two groups of treatment by 10% serum containing Dulbecco minimum essential medium (DMEM)/F12 medium (group A), or the same medium supplemented with 25% human vitreous (group B). The morphological changes were observed with a phase contrast microscrope. Cell migration, invasion and contractility were tested using a scratch wound assay, Transwell invasion assay and collagen gel contraction analysis. The expression levels of alpha;-smooth muscle actin (SMA) and Snail1 were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The RPE cells in group A were flat and gathered together. The RPE cells in group B grew as a fan-shaped projection at one edge and cone-shaped tail at the opposite edge, or spindle-shaped, and appeared to separate. In group A, filamentous actin distributed mainly at the margin of the cells with the distribution an oval shape. In group B, filamentous actin reorganized and formed fan-like flat pseudopodia at one edge of the cells. Compared to group A, the migration and invasion of the cells increased significantly (t=14.190, 22.630; P＜0.05), but contractility decreased remarkably (t=6.221, P＜0.05) in group B. Compared to group A, the expression level of Snail1 mRNA increased significantly (t=3.218, P=0.032), but the expression level of alpha;-SMA mRNA decreased (t=3.990, P=0.016). Conclusions Vitreous humor can induce the EMT of RPE cells. Increasing cell migration, cell invasion, and expression of Snail1 mRNA as well as up-regulated cellsprime; contractility and expression of alpha;-SMA mRNA may be the mechanism.
Objective To observe the autofluorescence （AF） manifestation in related lesions of periphery retinopathy.Methods Sixty eyes of 42 patients with periphery retinopathy underwent the examination of Optomap fundus photograph (200deg;) and fundus fluorescein angiography (FFA). The HRAⅡ melaninrelated nearinfrared fundus autofluorescence (NIA, excitation 795 nm) and lipofuscinrelated fundus autofluorescence (FAF, excitation 488 nm) were measured for all the patients. The AF was recorded with nine images per second, and then a final AF image with 55deg; view and 822times;768 pixel was generated by the HRA. AF images can be valuable or valueless if there was or was not visible blood vessels and related retinal tissues on the image. AF from lesion regions can be normal or abnormal fluorescence comparing to the normal vascular and retinal tissue AF. The abnormal fluorescence was divided into no AF, weak AF and b AF relative to the background grayscale. The grading consistency of abnormal fluorescence based on FAF and NIA examination was comparatively analyzed. Results Valuable AF images were captured in 53/60 eyes （88.33%）and valueless AF images were captured in 7/60 eyes （11.67%）. Among 53 eyes with valuable AF image, NIA showed normal fluorescence in 28 eyes （52.83%），abnormal fluorescence with sheetlike, dotshaped or stripped in 25 eyes （47.17%）; FAF showed normal fluorescence in two eyes （3.77%）, abnormal fluorescence with sheetlike, scattered along vessels or pigments in 51 eyes （96.23%）. Twentyfive eyes with abnormal fluorescence were observed both in two examinations, including same grades in 18 eye （72.00%） and different grades in seven eyes （28.00%）. Conclusion The AF manifestation with different levels exists in related lesions of periphery retinopathy.
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.
【摘要】 目的 觀察不同種培養基中重組人色素上皮衍生因子(rPEDF)融合蛋白的表達。 方法 將前期研究已構建的pET28aPEDF原核表達重組體轉化E.coli BL21大腸桿菌表達宿主菌,酶切鑒定陽性菌落后,分別在M9和LB培養基中用異丙基βD硫代半乳糖(IPTG,IsopropylbetaDthiogalactoside)誘導表達,SDSPAGE電泳檢測表達的PEDF蛋白, 美國ImagePro Plus 分析系統進行蛋白定量分析。結果 LB和M9培養基中均獲得相對分子質量約54×103的rPEDF融合蛋白。但LB培養基獲得的是rPEDF融合蛋白的包涵體,目的蛋白占總蛋白含量為21046%,M9培養基獲得的是可溶性的rPEDF的融合蛋白,目的蛋白占總蛋白含量的1231%。結論 不同種培養基中均有rPEDF 融合蛋白的表達。【Abstract】 Objective To observe the express of recombinant pigment epithelial derivative facto (rPEDF) in the different medium. Methods The pET28aPEDF was transformed into E.coli BL21. After the colonies were positive identification which were induced by IsopropylbetaDthiogalactoside in medium M9 and LB. The PEDF protein were detected by SDSPAGE and analyzed by American ImagePro Plus system. Results LB and M9 medium obtained the relative molecular mass about 54×103 rPEDF fusion protein. But LB medium obtained the inclusion bodys of rPEDF fusion protein，the purpose protein account for 21.046%；LB medium obtained the soluble rPEDF fusion protein，the purpose protein account for 12.31%. Conclusion The rPEDF protein was expressed in the different medium.
OBJECTIVE:To observe the effect of dexamethasone to intracellular free Ca2+ of frozen RPE cells.
METHODS:The cultured human RPE cells were frozen for 30s at --70deg;C. The RPE cells were loaded with Fura-2/AM and analyzed using a digital imaging microscopy system,the effect of dexamethasone to intracellular free Ca2+ was measured at a serial concentration of 40, 60,100,150,200mu;g/ml.
RESULTS:The concentration of intracellular free Ca in frozen human RPE cells was increased to 18.6%~29.8% by dexamethasone at concenlration of 40mu;g/ml~60mu;g/ml,while was decreased to 28.4%~35.2% at 150mu;g/ml~200mu;g/ml.
CONCLUSIONS:Effect of dexamethasone showed two aspects of effect to frozen
cultured human RPE ceils,that it was inhibitor at high concentration and stimulator at low concentration
（Chin J Ocul Fundus Dis,1997,13: 86-88）
Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells.Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamineTM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 μmol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcripti on factor E2F,and inhibit the proliferation of HRPE cells.(Chin J Ocul Fundus Dis，2004，20：182-185)
Objective
To study the effect of hypoxia on proliferation of cultured bovine retinal pigment epithelium (RPE) cells and expression of the antiapoptotic protein bcl-2.
Methods
The bovine RPE cells were cultured under normal and hypoxic chamber respectively. After 24 hours, the proliferation of RPE cells was evaluated by［3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide, MTT］test. At the same time, anti-bcl-2 protein antibody was examined by immuno-histochemistry method.
Results
The A value in the hypoxia group was higher than that in the normal group after 24 hours (P＜0.05 )in MTT-test. Positive staining for anti-bcl-2 protein antibody was seen in 72.6% cells in hypoxia group and 38.64% in normal group. The positive staining was more obvious near the nucleus, and fine granules scattered in cytoplasm of some cells.
Conclusion
Hypoxia can stimulate the proliferation of RPE cells and expression of antiapoptotic protein bcl-2. The results indicate that bcl-2 may play an important role in mediating the proliferation activity of RPE cells.
(Chin J Ocul Fundus Dis, 2002, 18: 293-295)
