Objective To explore a better method in obtaining iris pigment epithelium(IPE) specimen for autologous transplantation in rabbits． Methods IPE was obtained from 20 black rabbits with method A，i.e.surgical peripheral iridectomy at 12:00 position obtaining a triangle iris tissue with the hemline of 4-5 mm in left eyes，and method B,i.e.surgical peripheral iridectomy at 11:00 and 1:00 positions obtaining two triangle iris tissues with the hemlines of 2-2.5 mm in right eyes . The IP E cells were isolated precisely with enzyme microdissection-enzyme isolation method, cultured in vitro, observed with light and electronic microscope, and ident ified with immunocytochemical staining.ResultsThe success ra te of cells culture were 65% for method A and 95% for method B. After 3-4 generations of culturing，the amount of IPE cells was enough for transplantation, and most of the functions of primary clutured IPE cells were kept still. Viability of IPE cells was 85%-93%. Conclusion The success rate of cells culture for method B is higher than that for method A. The third generation of cultured cells is available for autologous transplantation.(Chin J Ocul Fundus Dis，2003，19：201-268)
Purpose
To investigate the effects of human vitreous fluid on proliferation of cultured human retinal pigment epithelial (RPE) cells and vascular endothelial cell lines(VEC304).
Methods
Human RPE cells and VEC304 were cultured and treated in different human vitreous-conditioned medium (VCM) with or without serum, vitreous volume concentrations of VCM were 1∶8, 1∶4 and 1∶2. Cells proliferation was assayed by tetrazolium (MTT) colorimetry at the 2nd, 4th and 6th day respectively.
Results
In the presence of serum, 1∶4, 1∶2 VCM had a significantly stimulative effect on RPE cells proliferation compared with control group at the 2nd, 4th, and 6th day retrospectively (P＜0.01), but exerted a bly inhibitory effect on VEC304 proliferation compared with control group at the 2nd, 4th, and 6th day retrospectively (Plt;0.05). In the absence of serum, only 1∶2 VCM had a stimulative effect on RPE cells growth compared with control group at the 2nd day (P＜0.05) and obviously at the 4th and 6th day respectively (P＜0.01).
Conclusion
Human vitreous fluid influences human RPE cells and VEC304 growth in vitro. This result suggests that vitreous may play different role in proliferative vitreoretinopathy and intraocular neovascular disease.
(Chin J Ocul Fundus Dis, 2002, 18: 140-142)
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 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.
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.
Objective
To investigate the effects of applied electric fields on the migration of human
retinal pigment epithelial (hRPE) cells, and probe the physiological effects and
clinic significance of electric fields on the RPE cells during the wound healing
process.
Methods
hRPE cells were clutured with Dubbeccolsquo;s modified Eagle medium (DMEM)
(DMEM group ) or DMEM+10% fetal bovine serum (FBS) (DMEM+10% FBS group), and the cells unexposed to the electric fields were used as the control. After hRPE cells were exposed to direct current electric fields with the tension of 0,4, 6 and 8 V/cm, serial images were taken at 15 minuets intervals within 2 hours by photomicroscope to observe the migration of the cells, and then the migrating distances and angle between the cellular translocation direction and the field vector were measured. The results were analyzed by an image analyzer. Resutls The response of the hRPE cells cultured in DMEM group to the electric fields was not as obvious as that in DMEM+10% FBS group. The cells showed a tendency of cath odal migration when the field tension was added to 6 V/cm (the average cosine Phi; was compared with the normal control, P＜0.05.The effect of electric
fields on cells clutured in DMEM+10%FBS group was ber than those in DMEM group. The long axes of the cells were perpendicular to the field line with the tension of 4 V/cm, and the cells continued migrating to the cathode, which was more obvious when the field tension increased (the average cosine Phi; was comparedwith the normal control, P＜0.05). After the polarity of the electric field reversed , the cells migrated to the opposite direction in accord with the direction of the cathode.
Conclusion
Applied electric fields can induce the cathode-directed migration of hRPE cells,
which is positively correlated with the field tension and can be influenced by the serum. Physiological electric fields may be one of the inducing factors of cell migration at the early phase of healing process of wounded retina.
(Chin J Ocul Fundus Dis, 2006, 22: 404-407)
OBJECTIVE:To investigate the transporting function of subretinal fluid in retinal pigment epithelium(RPE) of experimental retinal detachment (RD).
METHODS: Twenty-seven pigmented rabbits with one eye as retinal detachment model and the other eye as control were divided into 4 groups ,ic. 7 rabbits in each group at random. The Na十-K+-ATPase activity in RPE-choroidal tissue at the detached region in both experimental and control eyes was determined at the lst,2nd,3rd and 4th week after retinal detachment,and also the ultrastructure of the RPE-choroidal tissues observed. RESULTS:The
activity of Na十-K+-ATPase of experimental eyesE[(2.16士1.26)/mu;mol(mgmiddot;h)]was significantly lower than that of the controis[(4.84plusmn;1.59)mu;mol/(mgmiddot;h)]. The degree of decreasing activity was relieving along with increasing of the course of RD. The inward pinocytosis in RPE cells under electron-microscope was found from the 2nd week samples afterwards and becoming obvious along with increasing of the disease course.
CONCLUSIONS:The outward transporting function of RPE was increased after RD,it became decreased along with increasing of the course df RD and the inward transporting function then developed.
【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 investigate the effects of genistein with different concentration on proliferation and apoptosis of cultured human retinal pigment epithelial (RPE) cells in vitro. Methods The effect of genistein with the concentration of 5，10，25，50，75，and 100 mg·L-1on the proliferation of cultured RPE was examined by tetrazolium salt (MTT) assay and AgNORs staining. The cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) methods, in the mean time, the morphologic changes of cell apoptosis were observed by light microscopy and transmission electronic microscopy, the results of which were compared with the normal RPE cells. Results Genistein with the concentration of 25, 50, 75, and 100 mg·L-1had a dose-dependent and time-dependent antiproliferative effects on RPE cells with the inhibitory rate of 12.0%-64.6% (P＜0.05). The results of AgNORs staining showed that the number of AgNORs in the nucleolus decreased when treated by genistein. In TUNEL staining, the median of percent of apoptotic RPE cells was 7.6%, 9.8%, 13.7% when treated with 50 mg·L-1genistein, 10.3%, 16.4%, 23.4% when treated with 75 mg·L-1genistein, and 15.4%, 21.2%, 35.8% when treated with 100 m g·L-1genistein respectively for 24, 48, and 72 hours. After the treatme nt with 50 mg·L-1genistein for 48 hours, the apoptosis in the nucleolus of RPE cells was found. Conclusions Genistein with different concentrations has a dose-dependent and time-dependent antiproliferative effect on RPE cells. Genistein can induce the apoptosis of RPE cells when it reaches a certain extent of concentration. (Chin J Ocul Fundus Dis，2004，20：241-244)
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)
