Study On Relationships Of Phagocytosis, Expression Of MERTK Gene And Regulation By Protein Linase C In Human Retinal Pigment Epithelium Cells

ObjectiveThe phagocytic fuction of human retinal pigment epitheliumis ( HRPE) cells is crucial for sustaining the vision. The failure of phagocytosis will lead to the compiling of rod outer segments ( ROS) and seriers eye degeneration. Since the mechanism of phagocytosis is unknown by now, the further investigation of phagocytic mechanism will invest hopeness to the prevention and therapy of this kind of disease.The Royal College of Surgeons ( RCS) rat is a widely studied and classic model of recessively inherited retinal degeneration. Researches have revealed a null mutation , which lead to the failure of uptaking ROS during phagocytosis, in Mertk gene of RCS rats. Researches suggested the abnormal signaling transduc-tion in RPE of RCS rats too. For example, the disturbed modulation of L - Ca channel by tyrosine kinase and protein kinase C.These results didnt only highlight the critical role of receptor protein tyrosine kinase activity in the mechanism of RPE phagocytosis, but also brought forward some new problem for the further investigation in phagocytosis mechanism-----Whether protein kinase C as the signal messenge is involved in the phgocy-tosis of HRPE cells? How about the express level of MERTK mRNA in specific phagocytosis process? How MERTK and PKC react each other in the phgocyto-sis of HRPE cells? Herein, in this study, we set up the normal phagocytic conditions of HRPE cells, examined the phagocytic kinetics of HRPE cells .detected continuously the level of PKC during phagocytosis of HRPE , test the expres-sioq of MERTK gene mRNA in vivo, then studied the fuction of PKC in the phagocytosis process and clarified the relationship between protein kinase C and MERTK gene in the phagocytosis of HRPE cells .MethodsSection 1. Tissue culture of and fluorescent labeling of human retinal pigment epithelium cells . To collect the HRPE cells by the trypsin. The 3-5 passaged cells which reached 90% "Confluence were stained in growth medium containing SR before assay. ROS which was isolated by sucrose density gradient centrifugation was stained in the FITC suspension. Latex beads (LB) has fluorescent itself. Added FITC - ROS or LB each well to incubation HRPE cells at 37 °C. To terminate the phagocytosis by the time of 5min, 15min, 30min, 90min,3hours, 6hours, 12hours, 18hours, 24hours,36 hours and 48hours. To observe the binding and ingestion by HRPE cells under fluorescent microscope eqqiped with triple filter and long - distance appendixes. After 6 hours incubation of ROS/LB, the samples were fixed for scanning and transmission electron microscope. HRPE cells seeded in the special dishes were incubated with ROS for 30min or LB for 6hours respectively , then were observed under con - focal laser microscope.Section2. To incubate HRPE cells seeded in 12 - well plates with LB or unstained ROS. To collect the cells at different times as above and to examine the activity of PKC by methods of phosphorylation of substrate . To contrast the results with the control group.Section3. The examination of the express level of MERTK gene . To incubation HRPE cells with ROS at 37 °C and teminate reaction at different time (5min, lSmin^Omin, 9()min, 3h, 6h,12h, 18h, 24h ). The total RNA was extracted by RNA 0UT fliud . RT - PCR reaction contrasted by (3 - actin was conducted for examing the level of MERTK mRNA. The PCR productions were lo-ded in agar gel and scanned. The grey density were analyzed by Molecular Analyst (versionl.4.1).Section4. The relationship between MERTK gene , protein kinase C in thephagocytic process of human retinal pigment epithelium cellsTo treat the HRPE cells with different density of PMA /ST at 31X.. To exact the total RNA by RNA 0UT fliud . RT - PCR reaction contrasted by (3 - actin was conducted to investigate the dose - effect relationship between MERTK and PMA/ST . Pretreating the HRPE cells with lOOnM PMA /2.5um ST , ROS suspension was added to HRPE cells for further incubation (5min,15min, 30min, 90min,3h>6h,12h,18h>24hl respectively). Then RT - PCR reaction contrasted by (3 - actin was conducted again to clarify the effect of altered activity of PKC on the expression of MERTK. Statistics analysis;To deal with the data by of SPSS11.0 statistical software package. To com-parise the average numbers by Student 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 18h and 24h when the binding and ingestion were saturated respectively.(2)The nonspecific phagocytosis:The binding of LB by HRPE cells happened at 90 min, while the ingestion was detectable at 6 hours after feeding. The quantity of binding and ingestion were increasing with time during all the 48 h .2. The activity of PKC in HRPE cells during phagocytosis;(1 ) The specific phagocytosis;When HRPE cells were incubated with ROS, the activity of PKC ( both in cytoplasm and on membrane) decreased at 5 min,and it kept the steady lower level during all the incubation periods compared with control, and reached the minimum at 24h.(2) The nonspecific phagocytosis: When HRPE cells were incubated withLB, the activity of PKC did not changed in all the incubation time (5min -48h).3. At 5 min of incubation with ROS, the expression level of MERTK began to increase and kept the higher level than controls during all the incubation time ( from 5min to24h).4. After treated with PMA at different density, the expression of MERTK gene was significantly decreased in a dose - dependent manner . When HRPE cells which were pretreated with lOOnM PMA were incubated with ROS, the expression of MERTK was decreased in the proceeding incubation with ROS contrasted with control. After treated with ST at different density, the expression of MERTK gene was" increased without dose—dependent, manner . When HRPE cells which were pretreated with 2. 5 jxM ST were incubated with ROS, the expression of MERTK increased during the first 30min and decreased to the normal level at 90min of incubation.Conclusion1. The specific phagocytosis of HRPE cells has the characters of higher -effection and saturation,while nonspecific phagocytosis is unsaturated.2. The lower activity of PKC is very important fo sustaining the ingestion process in specific phagocytosis of HRPE cells, but PKC has no direct realation-ship with nonspecific phagocytosis .3. The higher expression level of MERTK is the key to the uptake process of ROS in HRPE cells.4. PKC seem to interact with MERTK by negative - correlation in the phag-ocytic process of ROS by HRPE cells .5. PKC and MERTK are two up - stream controller who regulate specific phagocitisis of HRPE cells by different signal transduction pathway.