【摘要】 目的 观察不同种培养基中重组人色素上皮衍生因子(rPEDF)融合蛋白的表达。 方法 将前期研究已构建的pET28aPEDF原核表达重组体转化E.coli BL21大肠杆菌表达宿主菌,酶切鉴定阳性菌落后,分别在M9和LB培养基中用异丙基βD硫代半乳糖(IPTG,IsopropylbetaDthiogalactoside)诱导表达,SDSPAGE电泳检测表达的PEDF蛋白, 美国ImagePro Plus 分析系统进行蛋白定量分析。结果 LB和M9培养基中均获得相对分子质量约54×103的rPEDF融合蛋白。但LB培养基获得的是rPEDF融合蛋白的包涵体,目的蛋白占总蛋白含量为21046%,M9培养基获得的是可溶性的rPEDF的融合蛋白,目的蛋白占总蛋白含量的1231%。结论 不同种培养基中均有rPEDF 融合蛋白的表达。【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.
ObjectiveTo observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress. MethodsThe third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier. The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10% normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro. hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group. Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours. Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment. TER was measured to evaluate the barrier function of hRPE. The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2), together with complement bioactive fragments (C3a, C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay. ResultsStable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert. Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51% compared with normal hRPE barrier. CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48% compared with normal hRPE barrier(t=21.60, P < 0.05). Compared with model group, CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48% and 23.47% secreted by hRPE under complement-activated oxidative stress (t=3.26, 2.43; P < 0.05). Compared with model group, CR1 treatment could also decreased the concentration of C3a, C5a and MAC by 24.00%, 27.87%, 22.44%.The difference were statistically significant (t=9.86, 2.63, 6.94; P < 0.05). ConclusionsCR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress. The underlying mechanism may involve inhibiting complement activation and down-regulating the expression of VEGF and CCL2.
ObjectiveTo observe the effect of subretinal injection of retinal pigment epithelium (RPE) cells for RPE in mice. MethodsA total of 30 postnatal day 7 C57BL/6J mice were randomly divided into normal mice group, OIR model group and OIR model cell transplanted group, 10 mice in each group. The OIR model was induced in mice of OIR model group and OIR model cell transplanted group. The RPE cells were subretinal injected into the RPE of mice in OIR model cell transplanted group. At 20 days after the injection, the RPE thickness was evaluated by fluorescence microscope. The expression of RPE65, Bestrophin and zonula occludens-1 (ZO-1) were estimated by Western blot and real-time quantitative PCR (RT-PCR). ResultsThe thickness of RPE in OIR model mice was thinner than that in normal mice; the thickness of RPE in OIR model cell transplantation mice was significantly thicker than that in the OIR model mice. The results of Western blot and RT-PCR indicated that the differences of protein (F=8.597, 18.864, 25.691) and mRNA expression (F=39.458, 11.461, 34.796) of RPE65, Bestrophin, ZO-1 were statistically significant between OIR model group and OIR model cell transplanted group (P < 0.05). ConclusionsSubretinal injection of RPE cells can promote RPE thickening. RPE65 and Bestrophin protein relative expression levels increased, ZO-1 protein relative expression levels reduced; mRNA expression levels of RPE65, Bestrophin and ZO-1 genes increased.
ObjectiveTo observe the clinical characterisitics of choroidal excavation in the macula. MethodsA total of 22 patients (22 eyes) with choroidal excavation diagnosed by spectral domain high definition optical coherence tomography (HD-OCT) were enrolled in this study. The patients included 12 males (54.50%) and 57 females (45.50%). The age was ranged from 21 to 82 years old, with an average of (41.44±13.17) years. All the patients were affected unilaterally, including 9 right eyes and 13 left eyes. The corrected vision, slit lamp microscope with preset lens, fundus photography, HD-OCT and fluorescence fundus angiography (FFA)were measured for all patients. The clinical characterisitics and concomitant diseases were observed. Seventeen eyes were followed for a period between 3 to 12 months. The lesions change were evaluated by HD-OCT. ResultsThere were 18 eyes (81.8%) with symptoms of micropsia and metamorphopsia, 4 eyes (18.2%) without symptoms. The corrected vision was ranged from 0.3 to 1.2, 12 eyes (54.54%) with moderate or high myopia. Fundus examination presents yellowish-white exudation in 12 eyes (54.54%), yellowish-white exudation accompanied with hemorrhage in 9 eyes (40.91%), grayish yellow reflex halo in 1 eye (4.55%). HD-OCT showed that the retinal pigment epithelium (RPE) layer was involved in the excavation, and the photoreceptor outer segment and pigment junction (OPR) layer was disappeared in all eyes. The external limiting membrane and the photoreceptor inner segment/outer segment junction layer were preserved in 13 eyes (59.09%) and disappeared in 9 eyes (40.91%). There were 10 eyes (18.18%) with a single lesion, 4 eyes (18.18%) with idiopathic choroidal neovascularization, 4 eyes (18.18%) with punctate inner choroidopathy, 1 eye (4.55%) with polypoidal choroidal vasculopathy, 1 eye (4.55%) with macular preretinal menbrance, 1 eye (4.55%) with central serous chorioretinopathy. FFA showed hypofluorescence in early phase, hyperfluorescence in late phase, without obvious leakage. There was no noticeable changes in size and morphological changes in the follow-up period. ConclusionsChoroidal excavation in the macula occurs mostly in middle-aged people with myopia. It can be associated with many fundus diseases. The excavation is located in RPE layer, and OPR layer disappeared. Choroidal excavation in the macula develops slowly.
Objective To investigate the effects of exosomes from cultured human retinal pigment epithelium (ARPE-19) cells affected by oxidative stress on the proliferation and expression of vascular endothelial growth factor-A (VEGF-A) and Akt of ARPE-19 cells. Methods Culture ARPE-19 cells. The concentration of 2.5 μmol/L rotenone was selected to simulate oxidative stress and isolated ARPE-19-exosome. Exosomes were isolated by ExoQuick exosome precipitation solution. Transmission electron microscopy was used to identify the morphology of exosomes. Western blot was used to detect exosomes’ surface-specific maker protein CD63. ARPE-19 cells affected by oxidative stress were cultured with exosome as experimental group, normal ARPE-19 cells were cultured with exosome as control group. The cell proliferation was examined by methyl thiazolyl tetrazolium assay. Western blot and immunofluorescence assay were used to detect the expression levels of VEGF-A and Akt protein. Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF-A mRNA and Akt mRNA. Results The diameter of normal ARPE-19-exosomes ranged from 50 to 150 nm. The isolated exosomes expressed CD63. AREP-19 cells were cultured with ARPE-19 (affected by rotenone)-exosome, the cell viability in experimental group was significantly reduced than in the control group. Green fluorescence was observed in the cytoplasm under fluorescence microscope. Compared with the control group, VEGF-A was up-regulated expressed and Akt was down-regulated expressed. Western blot results showed that, VEGF-A protein expression in the experimental group were higher than the control group. Akt protein expression in the experimental group were less than the control group. The difference was statically significant (t=3.822, 6.527;P＜0.05). RT-PCR results showed that VEGF-A mRNA expression levels was higher in the experimental group than the control group. Akt mRNA expression levels was lower in the experimental group than the control group. The difference was statically significant (t=8.805, −7.823;P＜0.05). Conclusions Exosomes from ARPE-19 cells affected by oxidative stress inhibit the proliferation of normal ARPE-19 cells, increase the expression of VEGF-A and reduce the expression of Akt.
ObjectiveTo observe the effect of TGF-β receptor inhibitor Compound C on the directed differentiation of human embryonic stem cells (hESC) into retinal pigment epithelial (RPE) cells. MethodsH1 hESC were divided into control group and experimental group. When the hESC reached over confluence, the medium was changed to knockout serum replacement medium without bFGF to induce RPE differentiation. The experimental group was supplemented with 1 μmol/L TGF-β receptor inhibitor Compound C at the first six days of induction. Real-time PCR was carried out to examine the expression of paired-box gene 6 (PAX6), microphthalmia-associated transcription factor (MITF), cellular retinaldehyde blinding protein (CRALBP), and RPE65 in both groups at the 1, 3, 5 weeks of the induction process. hESC-derived RPE (hESC-RPE) cells were isolated mechanically and purified. Real-time PCR, Western blot and immunofluorescence were used to characterize the purified hESC-RPE cells. ResultsPigmented colonies were observed in experimental group at the 4 weeks of the induction process, while no pigmented colony could be detected in the control group. All the purified pigmented cells from experimental group showed polygons morphology. Experimental group showed significantly higher expression of RPE marker genes PAX6, MITF, CRALBP and RPE65 than the control group(P<0.05). Compared with the hESC and ARPE-19 cells line, purified hESC-RPE cells showed much higher expression of PAX6, MITF, CRALBP and RPE65(P<0.05).High expression level of PAX6 and RPE65 proteins were observed in hESC-RPE cells. Immunofluorescence verified the expression of PAX6 and ZO-1 in hESC-RPE cells. ConclusionTGF-β receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE.
ObjectiveTo preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis (EAU).MethodsRPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/L adenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data.ResultsWhen the stimulation mode was AMP, the proliferation of CD4+ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the non-intervention group (F=13.28, P＜0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4+ T cells between the two groups (F=7.78, 6.58; P＞0.05). There was no statistically significant difference in the proliferation capacity of CD4+ T cells between the CD73-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P＞0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group (F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P＜0.01).ConclusionMMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.
Objective To investigate the protective effects of riluzole, a sustained activator of K2P subfamily member TRAAK potassium channel, in human retinal pigment epithelium (hRPE) cells with oxidative induce by tert-butyl hydroperoxide (t-BHP) in vitro, and to evaluate the possible involvement of K2P in the cytoprotective function of retina degeneration diseases. Methods The third to fifth passage of the primary cultured hRPE cells were used in the following experiments．hRPE cells were divided into seven groups: normal control group.t-BHP (300 mu;mol/L) group.t-BHP with riluzole (2, 5, 10, 20 mu;mol/L) group and riluzole (10 mu;mol/L) group. The apoptosis was measured by the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, annexinV/PI double staining flow cytometry. Changes of cells and nuclei morphology were observed under a phase contrast microscope and a fluorescence microscope after 4prime;, 6-diamidino-2-phenylindole (DAPI) staining. Immunofluorescence 1abelling was carried out to analysis the expression of TRAAK. Results After 24 hours incubation with 300 mu;mol/L t-BHP, the cells viability decreased to (58.7plusmn;12.2)% as compared to the normal control groups. The cell viability of t-BHP with riluzole group at different concentrations was higher than the t-BHP group, while 10 mu;mol/L riluzole showed maximally protective effect on hRPE death induced by t-BHP（t=4.84.P＜0.05）. Riluzole remarkably decreased pyknotic nucleus and cell swelling when compared with t-BHP group. Morphology of cells was fusiform with the uniform elliptic nuclei in normal and riluzole group. The Results of annexinV/PI double staining flow cytometry showed that ratio of normal cells were （97.6plusmn;1.3）%, （70.3plusmn;7.0）%, （86.9plusmn;5.2）%, （93.9plusmn;1.5）% in normal group.t-BHP group.t-BHP with riluzole group and riluzole group respectively. The ratio significant decreased in t-BHP group when it was compared with the other groups (t=7.53, 4.59, 6.49, respectively.P＜0.05). By contrast with normal group and riluzole group, the ratio of normal cells in t-BHP with riluzole group had no statistical significance（t=2.94, 1.91, respectively.P＞0.05）. Riluzole (10 mu;mol/L) also significantly decreased the ratio of early stage apoptotic cells from (25.50plusmn;8.02)% to (1.20plusmn;0.72)% in t-BHP injured groups （t=7.13，P＜0.05）. The ratio of early stage apoptotic cells significant decreased in t-BHP group when it was compared with the normal group and riluzole group （t=7.07, 5.94, respectively.P＜0.05）. By comparison with normal group and riluzole group, there are no statistical significance in t-BHP with riluzole group（t=0.06, 1.18, respectively.P＞0.05）. The mean gray values of TRAAK expression were 0.040plusmn;0.003, 0.041plusmn;0.001, 0.049plusmn;0.001, 0.055plusmn;0.001 in normal group.t-BHP group.t-BHP with riluzole group and riluzole group respectively. TRAAK density was significantly higher in t-BHP with riluzole group and riluzole group（t=7.40, 12.70, respectively.P＜0.05）. Conclusions Riluzole can protect hRPE cells against oxidative injury-induced cell death at early apoptosis stage. The mechanism may relate to that riluzole can promote the expression of K2P TRAAK potassium channel.
Objective To study the relationship between the expression of sonic hedgehog (Shh) and vascular endothelial growth factor (VEGF) in hypoxic human retinal pigment epithelial (hRPE) cells. Methods Cultured hRPE-19 cells (3rd - 6th generations) were used in this experiment. hRPE-19 cells were divided into three groups including the control group, the hypoxia experimental group (100 μmol/L CoCl2) and the inhibition group (pretreatment with 20 μmol/L cyclopamine 1 hour before hypoxia). After culturing for 4, 8, 12 and 24 hours, the mRNA level of Shh and VEGF genes in these cells were measured by fluorescence quantitative polymerase chain reaction, and the protein level of Shh and VEGF in the supernatants were measure by enzyme-linked immunosorbent assay. The relationship between the expression of Shh and VEGF was analyzed by Pearson correlation analysis. Results The control group expressed low levels of Shh and VEGF mRNA/protein. The expression of Shh and VEGF mRNA/protein in the hypoxia experimental group was significantly higher than that in the control group (F=178.364, 183.732, 77.456, 91.572; P＜0.01). The expression of Shh and VEGF mRNA in the inhibition group was significantly lower than that in the hypoxia experimental group (F=68.745, 121.834; P＜0.01). In the hypoxia experimental group, the expression of VEGF protein was positively correlated with the expression of Shh protein (r=0.942, P＜0.05); and the expression of VEGF and Shh mRNA was positively correlated (r=0.970, P＜0.01). However, there was no significant correlation in the expression of VEGF and Shh mRNA in the inhibition group (r=0.915, P＞0.05). Conclusion There is a positive correlation between the expression of Shh and VEGF in hypoxic hRPE cells.
ObjectiveTo observe the expression of hot shock protein 47 (HSP47) in pre-retinal membrane of proliferative vitreoretinopathy (PVR) and the influence of transforming growth factor-β2 (TGF-β2) on the expression of HSP47 in retinal pigment epithelial (RPE) cell. MethodsPre-retinal membranes were collected and observed by hematoxylin-eosin, Masson and immunohistochemical staining. Cultured ARPE-19 cells were treated with TGF-β2 at serial concentration (0, 1, 5, 10 ng/ml) and time (0, 12, 24, 48 hours), respectively. And then the mRNA and protein expressions of HSP47 and Col-Ⅰ were measured by fluorescence quantitative reverse transcription polymerase chain reaction and Western blot at the same time. ResultsA lot of epithelial cells with pigmental particles were observed in pre-retinal membranes of PVR, much accumulated collagen protein was observed in the specimens, and HSP47 positive expression was bserved in cytoplasm and stroma of most of the epithelioid cells. Compared with 0 ng/ml group, the expressions of HSP47 mRNA in ARPE-19 were up-regulated by 1.32, 2.35, 1.85 fold, significant differences were observed in all groups (F=27.21, P<0.05); the expressions of protein were up-regulated by 2.33, 2.89, 2.60 fold, significant differences were observed in all groups (F=39.78, P<0.05). The expressions of Col-Ⅰ mRNA were up-regulated by 1.29, 1.52, 2.11 fold, significant differences were observed in all groups (F=23.45, P<0.05); the expressions of protein were up-regulated by 1.18, 1.49, 2.11 fold and significant differences were observed in all groups (F=29.10, P<0.05). Compared with 0 hour group, the expressions of HSP47 mRNA were up-regulated by 1.56, 1.84, 2.86 fold in ARPE-19 cells stimulated by 5 ng/ml TGF-β2 for 12, 24 and 48 hours, and the differences were all significant (F=31.56, P<0.05); the expressions of protein were up-regulated by 2.08, 2.37, 2.80 fold, and the differences were all significant (F=49.18, P<0.05). The expressions of Col-Ⅰ mRNA were up-regulated by 1.57, 1.86, 2.78 fold and the differences were all significant (F=54.43, P<0.05), the expressions of protein were up-regulated by 1.38, 1.59, 2.16 fold and the differences were all significant (F=42.52, P<0.05). ConclusionTGF-β2 may play a role in the pathologic process of PVR by promoting the expression of HSP47 and then increasing the synthesis and accumulation of Col-Ⅰ.