Objective To investigate the role of intracellular Ca2+ and MERTK in the phagocytosis of human retinal pigment epithelial (RPE) cells, and reveal the relationship between MERTK and intracellular Ca2+. Methods The cultured RPE cells were incubated with rod outer segments (ROS) at 37℃, the phagocytosis was terminated at different incubation time points. The concentration of intracellular Ca2+ was assayed by Fluro-3/AM loading methods combined with fluorescence microscope and CCD system, and the mRNA level of MERTK gene was measured by reverse transcription polymerase chain reaction (RT-PCR). Treating the RPE cells with stimulator (A23187) or inhibitor (verapamile) of intracellular Ca2+ to observe the changes of MERTK gene expression. Results ROS adhered to hRPE cells at the 15th minute, and the ingestion saturated at the 24th hour. The concentration of intracellular Ca2+ increased at the 15th minute, and kept the high level in 24 hours. The level of MERTK mRNA increased at the 5th minute, and kept the high level duration the whole incubation. When RPE cells were treated by A23187, the expression of MERTK increased in a dose-dependent manner. After RPE cells was pretreated by A23187, the expression level of MERTK was higher in the proceeding incubation groups than which in the control group except at the 3rd hour. When RPE cells were treated by verapamil, the expression level of MERTK decreased in a dosedependent manner. After RPE cells were pretreated by verapamil , the expression level of MERTK was lower in all the proceeding incubation groups than which in the control group (Plt;0.05). Conclusion MERTK gene and Ca2+ play an important role in sustaining RPE cells phagocytizing ROS. As an up-stream regulator, the receptor tyrosine kinase MERTK keeps RPE cells phagocytizing ROS by starting the intracellular Ca2+.
ObjectiveTo observe the concentration of the inflammatory cytokines in vitreous of severe proliferative diabetic retinopathy (PDR) after intravitreal ranibizumab injection (IVR). MethodsA total of 80 PDR patients (80 eyes) were enrolled in this study. The patients were randomly divided into vitrectomy group (group A) and IVR combined with vitrectomy group (group B), 40 eyes in each group. The differences of sex (χ2=0.05), age (t=0.59), duration of diabetes (t=0.36), HbA1c (t=0.13) and intraocular pressure (F=0.81) between two groups were not significant (P>0.05). The eyes in group B received 0.5 mg (0.05 ml) ranibizumab injection at 7 days before operation. The vitreous samples (0.4 ml) were obtained before operation. The concentration of vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, intercellular adhesion molecule-1 (ICAM-1) and connective tissue growth factor (CTGF) were measured by enzyme-linked immunosorbent assays. ResultsThe concentration of VEGF and ICAM-1 were (10.70±3.60), (224.64±90.32) pg/L in group B and (72.38±23.59), (665.61±203.34) pg/L in group A. The differences of VEGF and ICAM-1 concentration between two groups was significant (t=16.34, 12.53; P<0.001). The concentration of IL-6 and IL-8 were (210.64±80.27), (156.00±57.74) pg/L in group B and (45.78±33.82), (41.07±13.82) pg/L in group A. The differences of IL-6 and IL-8 concentration between two groups was significant (t=11.97, 12.24; P<0.001). There was no difference of CTGF concentration between two groups (t=1.39, P=0.17). The CTGF/VEGF in group B was higher than that in group A (t=14.75, P<0.001). ConclusionsOne week after IVR, the concentration of VEGF and ICAM-1 are decreased, while IL-6 and IL-8 increased. There is no obvious change in CTGF, but CTGF/VEGF is increased.