Mertk gene expression and photoreceptor outer segment phagocytosis by cultured rat bone marrow mesenchymal stem cells.

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSCs) are multipotential stem cells that have been used for a broad spectrum of indications. Several investigations have used BM-MSCs to promote photoreceptor survival and suggested that BM-MSCs are a potential source of cell replacement therapy for some forms of retinal degeneration. PURPOSE: To investigate the expression of the MER proto-oncogene, tyrosine kinase (Mertk), involved in the disruption of RPE phagocytosis and the onset of autosomal recessive retinitis pigmentosa in rat BM-MSCs and to compare phagocytosis of the photoreceptor outer segment (POS) by BM-MSCs and RPE cells in vitro. METHODS: MSCs were isolated from the bone marrow of Brown Norway rats. Reverse transcription-PCR (RT-PCR) and western blot analyses were used to examine the expression of Mertk. The phagocytized POS was detected with double fluorescent labeling, transmission electron microscopy, and scanning electron microscopy. RESULTS: Mertk expression did not differ among the first three passages of BM-MSCs. Mertk gene expression was greater in the BM-MSCs than the RPE cells. Mertk protein expression in the BM-MSCs was similar to that in the RPE cells in the primary passage and was greater than that in the RPE cells in the other two passages. BM-MSCs at the first three passages phagocytized the POS more strongly than the RPE cells. The process of BM-MSC phagocytosis was similar to that of the RPE cells. CONCLUSIONS: BM-MSCs may be an effective cell source for treating retinal degeneration in terms of phagocytosis of the POS.

Curative effect assessment of bandage contact lens in neurogenic keratitis.

AIM: To observe the curative effect of bandage contact lens in neurogenic keratitis. METHODS: Twenty cases of neurogenic keratitis were studied at the Department of Ophthalmology, the first Affiliated Hospital of China Medical University, between October 2012 and June 2013. These included 13 males and 7 females, aged from 35 to 88y. Patients were voluntarily divided into an experimental group (lens wearing group, n=10) and control group (drug therapy, n=10). In experimental group patients wore silicone hydrogel bandage soft contact lens. Both groups used the following eyedrops: 0.5% levofloxacin TID; 0.5% Sodium carboxymethyl cellulose QID; fibroblast growth factor BID; ganciclovir BID [cases complicated with herpes simplex virus (HSV)]; compound tropicamide BID (cases concurrent hypopyon). The healing time of corneal ulcer and complication rates were observed in the two groups. RESULTS: The healing time of corneal ulcer in the experimental group was 10.80±4.44d versus 46.70±13.88d in the control group (P<0.05). No complications occurred in the experimental group, except for the lens falling off twice in one case, the patient recovered eight days after rewearing the lens. While in the control group, all cases vascularized, 2 cases were complicated with descemetocele that recovered with amniotic membrane transplantation and 1 case was complicated with corneal perforation that recovered by autologous conjunctival flap covering. CONCLUSION: Bandage contact lens is a safe and effective method of treating neurogenic keratitis and significantly shortened the healing time of corneal ulcer.

[The relationship between MERTK gene and protein kinase C in the phagocytic process of human retinal pigment epithelial cells].

OBJECTIVE: To investigate relationship of the expression of MERTK gene and the activity of protein kinase C (PKC) in the phagocytic process of human retinal pigment epithelial (hRPE) cells. METHODS: Cultured hRPE cells were incubated with rod outer segments (ROS) suspension (containing ROS 1x10(10)/L) at 37 degrees C, then cells were rinsed at different times to terminate the phagocytosis. The kinetic of phagocytosis was measured by double-fluorescent labeling. The activity of PKC and the expression level of MERTK gene were measured by counting gamma-32P radio-activity with liquid scintillation and RT-PCR respectively. Change of MERTK gene expression was measured after hRPE was treated cells with stimulator or inhibitor of PKC. Statistical analysis was performed by SPSS 13.0 software. RESULTS: The phagocytic assay showed that the quality of bound and ingested ROS by hRPE cells increased. The quality of ingested ROS by hRPE cells at 24 hours was (2.85+/-0.11)x10(6), which reached maximum contrast with control (0.00+/-0.00)x10(6) (t=47.64, P<0.05). The activity of PKC (both in cytoplasm and on membrane) decreased during all the incubation periods compared with control [cytoplasm: (329.63+/-14.26) nmolxg(-1)xmin(-1)and on membrane: (467.67+/-68.87) nmolxg(-1)xmin(-1)], and reached the minimum at 24 h[cytoplasm: (151.13+/-17.67) nmolxg(-1)xmin(-1) and on membrane: (152.45+/-64.83) nmolxg(-1)xmin(-1); cytoplasm t=89.66 and membrane t=10.31, P<0.05]. The level of MERTK mRNA increased in pulse-chase and long-time incubation test. The gray level for 90 min was 1.8853+/-0.0077, contrasted with control 0.7246+/-0.0062, F=16,060.2167 and P<0.05. The gray level for 24 h was 0.5946+/-0.0082, contrasted with control 0.3343+/-0.0064, F=919.8421 and P<0.05. When up-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA was decreased in the proceeding incubating with ROS contrasted with control (pulse-chase group F=17,142.2331, long time group F=1886.4614; P<0.05). After down-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA waved between 4.4670+/-0.0092 and 5.7034+/-0.0095 in the first 30 min of incubating with ROS, which lower than control 0.9117+/-0.0021 (F=199,012.9138, P<0.05). CONCLUSIONS: The lower activity of PKC and the higher expression MERTK gene are very important for sustaining phagocytic process of ROS by hRPE cells. MERTK gene and PKC both as up-stream regulators are negative-correlated in the phagocytic process of hRPE.

[The alteration of cyclic adenosine monophosphate and protein kinase C in the phagocytic process of human pigment epithelial cells].

OBJECTIVE: To investigate the role of cyclic adenosine monophosphate (cAMP) and protein kinase C (PKC) in the phagocytic process of human retinal pigment epithelial (HRPE) cells by measuring phagocytosis indexes in specific and nonspecific phagocytic process. METHOD: The cultured HRPE cells were incubated with 1 x 10(7)/ml rod outer segments (ROS) or latex beads (LB) at 37 degrees C, then the phagocytosis were terminated at different incubation time (5 min-48 h). The kinetics of phagocytosis was measured by double-fluorescent vital assay. The binding and ingestion of ROS or LB were proved by scanning and transmission electron microscope. cAMP level was measured using (125)I-cAMP radio-immunity kit, and PKC activity was measured by counting gamma-(32)P radio-activity with liquid scintillation. RESULT: In specific phagocytosis of HRPE cells, the binding of ROS was happened at 15 min of incubation, and the cAMP level was decreased at the same time; the activity of PKC (both in cytoplasm and membrane)was decreased at 5 min which was earlier than the changes of the level of cAMP, but both indexes were reached their lowest level at 24 h. In nonspecific phagocytosis of HRPE cells, the binding of LB was happened at 1.5 h; on the other hand, when HRPE cells were incubated with LB, cAMP level did not change until 12 h (P > 0.05), but it began to decrease in the incubation time from 12 h-48 h; the activity of PKC (both in cytoplasm and on membrane) was stable during all incubation periods (P > 0.05). CONCLUSION: cAMP and PKC are very important for sustaining the ingestion process in specific phagocytosis of HRPE cells, but have no direct relationship with nonspecific phagocytosis. Furthermore, cAMP level and PKC activity in HRPE cells are decreased during specific phagocytosis.