1.1 Study subjects
We selected 15 patients with wet AMD (wAMD) and 16 age-and gender-matched normal people. Inclusion criteria: (1) AMD patients meet the diagnostic criteria in the Guidelines for the Diagnosis of Age-Related Macular Degenesis;9 (2) Eye examination and physical indicators in the control group were normal, with no family history of eye disease. Exclusion criteria: (1) patients with diabetes and severe cardiovascular and cerebrovascular diseases; (2) patients with cardiac, liver, renal impairments; (3) patients with other retinal diseases, glaucoma, etc; (4) patients with related eye treatment in nearly 1 month. This study was approved by the Ethics Committee of the Affiliated Hospital of Southwest Medical University.
1.2 Cell separation and culture
10ml of the blood of AMD patients and normal people were extracted into the heparin anticoagulant centrifuge tube and immediately mixed to avoid blood coagulation. Human peripheral blood mononuclear cells were obtained by centrifugation at a temperature of 4℃ and at a centrifugal force of 800g for 30 minutes after adding the human lymphocyte separation fluid. Then a 20ul monocyte magnetic beads (CD14 + magnetic beads (Miltenyi Biotec, Germany)) was added and incubated in a 4℃ freezer for 15min. The CD14+T cells were obtained by centrifugation of PBMCs with CD14+ magnetic beads on MS column for 1000g at room temperature for 5min. The CD14+T cells were added to the 1640 medium containing 1% glutamine, 1% penicillin streptomycin and 10% fetal bovine serum (Gibco BRL, USA). GM-CSF (100 ng/mL, ACRO Biosystems, Newark, USA) and People Regroup IL-4 (50 ng/mL, ACRO Biosystems, Newark, USA) were added into the medium to induce the formation of dendritic cells (DCs). After 6 days of cell culture, adding LPS (100ng/ml, Sigma, St. Louis, Missouri, USA) to cultivate for 24h to promote its transformation into mature dendritic cells.
1.3 Extraction and real-time quantitative PCR of RNA
According to the instructions, total RNA of PBMCs and DCs was extracted using Trizol lysate (Invitrogen, Carlsbad, California, USA) and cDNA was synthesized by reverse transcription (Takara, Tokyo, Japan). Synthetic conditions for real-time quantitative PCR were 95 °C pre-degeneration 30 seconds, 95 °C degeneration 5 seconds, 60 °C annealing 34 seconds, and 95 °C extended 15 seconds for a total of 40 cycles. In this experiment, the sequences of TNFAIP3and β-actin were as follows: TNFAIP3 forward: 5'-TGGCTGAACAAGTCCTTCCT-3′, reverse: 5'-CTTCAGGTCACCAAGGGTA-3′; β-actin forward: 5'-CGAGAAGATGACCCAGATCATG-3', reverse: 5'-CAGAGGCGTACAGGGATAGCA-3'. The data analysis is calculated using the 2- △ △ CT method.
1.4 Transfection of dendritic cells
Mononuclear-induced dendritic cell culture until the third day, inoculated to 24 orifice plates with a cell concentration of 1 × 105/ ml, and added the Human TNFAIP3 gene interferes with the adenovirus (Hanbio, Shanghai, China) with virus volume MOI of 10, 30, 100, 300, 500, another group of target cells was added with the same titer and volume of control virus vector, and changed the fluid after 2 hours. The expression of GFP green fluorescence was observed under the fluorescence microscope 48 hours after transfection.
To detect the mechanism of TNFAIP3 action in DCs, 48 hours after we transfected DCs with adenovirus, subsequently added the LPS (100 ng / ml) action for 30 minutes, and finally detected the phosphorylation levels of JNK, ERK1/2 and p38 (BD Biosciences, San Diego, California, USA) via flow cytometry.
Test the levels of IL-1β, IL-6 and IL-10 in the cell supernatant in accordance with the ELISA Kit Instructions (R&D Systems, Minneapolis, Minnesota).
1.7 Statistical analysis
All data were analyzed by SPSS17.0 software statistical analysis, expressed in the form of Mean土SD, normality tests using Shapiro-Wilk normality test, independent Samples T-test, and p <0.05 was seen as having a significant statistical difference.