| ObjectiveThe phagocytic faction of human retinal pigment epitheliumis ( HRPE) cells is crucial for sustaining the vision. The failure of this phagocytosis will lead to the compiling of rod outer segments ( ROS) and seriers eye degeneration. For the mechanism of phagocytosis is unknown by now, to further the investigation of phagocytosis, will invest hopeness to the prevention and therapy of this kind of disease.Though previou research has proved that the specific phagocytosis of RPE cells involves the interaction of ligment - receptor and trans-membrane signal conducting system, the fuction of transmembrane signal conducting system in the process of phagocytosis of HRPE cells is not very clear. Just for this reason , we chose cAMP which is one molecule of the signal conducting pathway as the study object to question the relationship between this signal molecule and the phagocytic fuction of RPE cells deeply, by modeling the phagocytic condition of normal HRPE cells in vivo and detecting the concentration of cAMP during the different stage in phagocytosis on the basis of dynamic observation of specific and nonspecific phagocytic character .Material and method1. The source of eye ballThe eye balls are the normal eyes which are after cornea transplantation in department of Ophthalmology of the First Clinical College of China Medical University. The donor of the eyes is limited between 24 and 38 ages.2. Tissue culture of and fluorescent labeling of human retinal pigment epithelium cellsTo collect the HRPE cells by the trypsin. The 3-5 passaged cells which reached 90% confluence were maintained for 36 hours before assay in growth medium containing SR at a final concentration of 40ug/ml.3. Isolation and fluorescent labeling of ( ROS )Isolated ROS by sucrose density gradient centrifugation and added the FITC stock to the resuspended ROS at a final concentration of 10ug/ml,and incubation was continued for 1 hour at room temperature , then diluted ROS to a final concentration of 1 × 107 ml-1 with growth medium.4. Preparation of latex beads ( LB )Diluted LB to a final concentration of 1 × 107 ml-1 with growth medium.5. Fluorescent observation the phagocytic kineticsAdded 1 × 107 ml "'FITC - ROS or LB 2ml/weU (6 well plate) to incubation HRPE cells at 37℃. Discarded the incubation fluid by time of 5min, 15min, 30min, 1. 5hour, 3hours, 6hours, 12hours, 18hours, 24hours, 36hours and 48hours,and washed the cells withDMEM at 4℃ twice toterminate the phagocytosis. By double fluorescent assay ,used the selective and nonselective barrier filter to observe the binging and ingestion by HRPE cells during different time of incubation.6. Scanning and transmission electron microscopyAfter 6 hours incubation ,the 2. 5 % glutaraldehyde was added to the cells to prepare the samples.7. Measuring of the concentration of cAMP in HRPE cells Experiment group: Added 1 ℃ 107 ml-1 ROS or LB 1 ml/well (24well plate) ; Control group: Added 1 ml DMEM + 10% FCS to each well. The time and method of terminating phagcytosis are same to a-bove. After terminating the phagocytosis,200ul/well DMEM (ph =7. 4) were added to HRPE cells, then boiled the cells 3min. Take out 100ul of each well for 125I - cAMP radio - immunity kit measurment.8. Statistics analysis:Each phagocytosis assay was repeated at least three times . Mean and standard deviation (x±s ) were calculated. Comparison between two samples were performed using Student* s t test. P <0.05 was seen as apparent discrepancy.Result1. The kinetic studies revealed that :(1) The specific phagocytosis:The binding of ROS by HRPE cells happened at 15 min after feeding with FITC - ROS, while the ingestion detectable as early as 30min . The quantity of binding and ingestion were increasing with time until 18 hours and 24hours when the binding and ingestion weresaturated respectively.(2)The nonspecific phagocytosis:The binding of LB by HRPE cells happened at 1. 5h, while the ingestion was detectab...
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