Experimental Study On Human Retinal Pigment Epithelium Cells' Proliferation Activity Following Vitreous Conditional Medium Treatment And The Mechanism

Experimental study on human retinal pigment epithelium cells' proliferation activity following vitreous conditional medium treatment and the mechanismObjectiveProliferative vitreoretinopathy (PVR) is such a proliferative disease including many cells, especially retinal pigment epithelium (RPE) cells, proliferation, migrating, and forming proliferative membrane in sub-or pre-retina or vitreous cavity. Unfortunately, the mechanism for it was still unknown, though there had been many study on the proliferation of RPE cells by growth factors and cytokines.The contact between RPE cells and vitreous following the injury of cerebral stratum of retina was a distinct risk of PVR, though the effect of vitreous was still uncertain. The quantity of cytokins had changed in the patho-process of PVR. Vitreous from patient of PVR can promote sythesis of collagenes and contraction of prolerative membrane, which participated in PVR in different link. Such results hinted that vitreous as a promoter played a crucial role in RPE cells' growth and PVR. We observed the changes in growth state of RPE cells and transcription level of c-fos and expression level of p-ERK followed by treatment of vitreous conditional medium. We also investigate the mechanism of these changes, which clarified the etiological reason of PVR and provided elementary rationale for clinical therapy.MethodsSection 1. The investigation of growth activity and cell cycle of HRPE cells following vitreous conditional medium treatment1. Cultivation and identification of HRPE cellsPrimary and serial subcultivation of HRPE cells were collected by trypsin under germ free condition. Pan-cytokeratin antibody was used to certify their epithelial origin.2. Preparation of vitreous conditional mediumVitreous, retina and pigmental tissue removed, was collected in 15mL centrifuge tube under germ free condition. Then was centrifuged at a rate of 10000r per minute, 0℃, filtered by membrane with 0.22μm millipore and stored in profound hypothermia refrigator at—78℃. VCM with different concentration (10%, 25%, 50% and 100%) were made by mixture of 1% FCS, DMEM and different volume of vitreous.3. HRPE cells' morphological observation, proliferative activity and cell cycle detection following VCM treatmentMorphological changes, proliferative activity and cell cycle of HRPE cells were pictured under inverted microscope and detected by MTT assay and Flow Cytometry respectively.Section 2. The examination of p-ERK in HRPE cells after VCM treatment1. Immunofluorescence examinationThe third passage HRPE cells were cultivated in 24-well plates. 25% VCM was added to experimental group, which was replaced by BSS in control group. The experimental was ended at different observing time. p-ERKs' distribution and expression was pictured under inverted fluorescence microscope.2. The expression level of p-ERK in HRPE cells following VCM treatment with different concentration or different observation time were detected by Western blot The third passage HRPE cells were cultivated in 75mL flask. HRPE cells were treated by different concentration VCM for 10min or 25%VCM for different durations in experimental group, VCM was replaced by BSS in the control group. The cells about 2×10⁶ were collected and western blot was used to detect the expression level of p-ERK in HRPE cells.Section 3. The examination of c-fos transcroption level in HRPE cells after VCM treatment1. Immunofluorescence examinationThe third passage HRPE cells were cultivated in 24-well plates. 25%VCM was added to experimental group, which was replaced by BSS in control group. The experimental was ended at different observing time. c-fos expression was pictured under inverted fluorescence microscope.2. The transcription level of c-fos mRNA in HRPE cells following VCM treatment with different concentration or different observation time were detected by RT-PCRThe third passage HRPE cells were cultivated in 75mL flask. HRPE cells were treated by different concentration VCM for 15min or 25%VCM for different durations in experimental group, VCM was replaced by BSS in the control group. The cells were collected and RT-PCR was used to detect the transcription level of c-fos mRNA in HRPE cells.Section 4. The signal transduction of c-fos in HRPE cells after VCM treatmentThe ca²⁺, PKC and ERK signal system were blocked by specific blocking agent then c-fos mRNA transcription level was detected by RT-PCR after 25% VCM added for 15min. Results1. The investigation of growth activity and cell cycle of HRPE cells following vitreous conditional medium treatment(1) Cultivation and identification of HRPE cellsThe just digested HRPE cells were round, and contained large quantity of pigments, which decreased along with the cell division. They propagate at a high rate and reached to confluent after 5 to 7 days. HRPE cells' origin were confirmed by positive pan-cytokeration immunocytochemical stain in cytoplam.(2) HRPE cells' morphological observation following VCM treatmentIn control group, primary passage HRPE cells showed a flat and irregular polygon, which can seen monocaryon or dikaryon, surrounded by many pigment granules. After treated by 10%, 25%, 50% VCM, HRPE cells growed at a higher rate, mitotic figure, even polynucleation was obviously found and depigmenting earlier, which showed fibroblast cell-like earlier too.(3) Proliferative activity observation following VCM treatmentHRPE cells in test group proliferated at a higher rate, which had significient difference in statistics analysis. The difference began at the third day, and last to the seventh day. 25%VCM had the most powerful effect in the HRPE cells' proliferation. Cells became apoptosis in 100% VCM.(4) Cell cycle detection after treatment of VCMHRPE cells showed a higher percent in S phase in experimental group, and the percent of G1, G2 phase was also changed at the same time. The most important outcome of us was that some heteroploid were found in 25% and 50% VCM group, which was obviously different from diplontic in control, 10% and 100% VCM group. 25% VCM had the most powerful effect in the happening of HRPE cells' heteroploid.2. The examination of p-ERK in HRPE cells after VCM treatment(1) Immunofluorescence examination 25% VCM promoted the translocation of p-ERK. In the control group, p-ERK distributed in cytoplasm, feeble stained, and the nucleus was unstained. After treatment of 25% VCM, the stain strength in cytoplasm was improved and translocated to nucleus, then reached to the rest state after lasting for about 2h.(2) The expression level of p-ERK of HRPE cells following VCM with different concentration or different observation time were detected by Western blotOur results was that the expression level of p-ERK had dose and time dependent effect with VCM. The expression was found as early as 5min, reached to maximum at 10min and lasted for 2 hours. 25% VCM had the most powerful effect in the expression level of p-ERK, 100% VCM was invalid in p-ERKs' expression.3. The examination of c-fos expression level in HRPE cells after VCM treatment(1) Immunofluorescence examination25% VCM promoted the expression of FOS. In the control group, FOS was feeble stained, and the nucleus was unstained. After treatment of 25% VCM for 10min, the stain in partial nucleus was found, and nearly all of nucleus were positive stained at 20min, then reached to the rest state after lasting for 60min.(2) The transcription level of c-fos mRNA of HRPE cells following VCM with different concentration or different observation time were detected by RT-PCROur results was that the expression level of c-fos had dose and time dependent effect with VCM. The c-fos mRNA transcription was found as early as 5min, reached to maximum at 15min, and lasted for 1 hour. 25% VCM had the most powerful effect in the expression level of c-fos.4. The signal transduction of c-fos in HRPE cells after treatment of VCMOur data showed that ca²⁺ had no obviously effect in c-fos trancription. But PKC and ERK signal system were participate in c-fos mRNA transcription. And the ERK activation in HRPE cells was not PKC-dependent.Conclusion1. HRPE cells showed changes in morphology, growth activity and cell cycle following vitreous conditional medium treatment.2. ERK in HRPE cells was activated after VCM treatment and translocated from cytoplasm to nucleus. The expression level had relationship between duration and concentration of VCM.3. The transcription of c-fos protooncogene was changed by VCM and the transcription level had some relationship between duration and concentration of VCM.4. There was no obviously relationship between ca²⁺ and transcription of c-fos protooncogene.5. PKC and MAPK signal transduction participated transcription of c-fos protooncogene.6. ERK activation in HRPE cells by VCM was not PKC-dependent.