1.1 Collection of clinical specimens
All patients were fully informed of the study and signed consent forms were provided.Patients with cancer, autoimmune diseases and other serious systemic diseases were excluded.This study followed the guidelines of Helsinki ethics committee and was approved by the ethics review committee of ophthalmic hospital affiliated to Nanchang University.Vitreous samples were collected during vitrectomy,Pvr-a (n = 3), pvr-b (n = 3), pvr-c (n = 3), pvr-a (n = 3), pvr-c (n = 3), pvr-a (n = 3), pvr-b (n = 3), pvr-c (n = 3).In the control group, the vitreous bodies of patients with corneal donation were collected (n = 3).The use of human tissue materials is in accordance with the Helsinki Declaration on the use of human body materials for research.
1.2 Cell culture and treatment
ARPE-19 was obtained from Shanghai Yubo Biotechnology Co., Ltdand cultured in DMEM / F12 medium (01-172-1acs, biological industries) containing 10% FBS (04-001-1acs) and 1% PS, and incubated in thermo (USA) at 37 ° C and 5% CO2 saturation humidity.24 hours later, the cells adhered to the wall completely.The cells were cultured to 80% − 90% fusion degree, starved overnight in serum-free medium, and replaced with DMEM / F12 containing 1% FBS,Different concentrations of artesunate (Solarbio) (0,50,100,150,200µCell proliferation and migration were detected at different time points (0, 24, 48 and 72 h).TGF was used-βThe degree of EMT was detected after 48 hours of pretreatment with artesunate.
1.3 Cell cycle was detected by flow cytometry
The cells were digested with trypsin (EDTA) and centrifuged at 350 g for 5 minthe supernatant was discarded.Cells were collected, washed with cold PBS, resuspended with 1 ml cold PBS, centrifuged for 5 min with 350 g, and the supernatant was discarded.Add 1 ml ice cold 75% ethanol, mix, incubate overnight at 4 ℃.500 g, centrifuged for 5 min, and the supernatant was discarded;The cells were then resuspended with 1 ml cold PBS and centrifuged at 500 g
5min, discard the supernatantThe sample was completely resuspended, added with 0.5 mL PI solution, and incubated for 25min at room temperature in the dark for flow cytometry analysis (inExcitation wavelength and emission wavelength of 615 nm detect red fluorescence signal)Cell fragments and aggregated cells were removed by flow cytometry, and DNA content of single living cells was analyzed by Modifit LT5 software.
1.4 Cell contraction assay
A cell contraction detection kit (cell Biolabs, San Diego, CA, USA) was used to evaluate the contractility of ARPE-19ARPE-19 (2×106 cells/ml) was suspended in basic medium, mixed with collagen gel, put in 24-well plate, and placed in incubator at 37 C and 5% CO2 saturation humidity for 1 hAfter gel polymerization, 1ml mixture of cells and collagen was addedAfter 48 h of cell culture, collagen gel was gently released from the sideAt last, Image-J(NIH, Bethesda,MD, USA) to image and quantify the size change of collagen gel (relative to the initial collagen gel size).
1.5 ELISAs
ARPE cells were treated with different concentrations of artesunate (0,75,150,200 µ mol/L) for 48 hoursCollecting cell culture solution, centrifuging to remove impuritiesELISA(༟) Pre-prepared reagents were used: diluted human TGF-β2 reference substance, biotin-labeled anti-TGF-β2 antibody working solution, avidin peroxidase complex (ABC) working solution.Add the standard substance and each group of samples into the ELISA plate in a volume of 100 µL/ well, seal the plate with membrane, and incubate at 37℃ for 90minShake off the liquid in the ELISA plate, do not wash it, add biotin-coupled anti-human TGF-β2 antibody, the workload is 100 µL/ well, and incubate at 37℃ for 60min after sealing the membraneThen, 300 µ L of 1× washing solution was added to each hole, and washed for 3 times, each time for 1minAdd ABC working solution into 100 µL/ well,The plates were sealed with membrane and incubated at 37℃ for 90minAdd 1× washing solution (300 µL/ hole) and wash for 5 times, each time for 1minAdd TMB chromogenic solution 90 µL/ well, and incubate at 37℃ in the dark for 15minWe added TMB termination solution at 100 µL/ mouth (in the same order as TMB)With enzyme-labeled analyzer, the OD value was measured immediately at 450nm wavelength, and the sample concentration was calculated by using the OD value as a standard curve.
1.6 Western blotting assays
The cells and tissues were washed twice with ice-cold phosphate buffer (PBS, 02-024-1acs, Biological Industries, Israel), added with protease inhibitor (PMSF, AR1178, Boster) and centrifuged at 12000 rpm for 15 minutesCollect supernatant lysate for BCA protein detection (BCA protein quantification kit, e112-01/02, Nanjing nuoweizan biotechnology co., ltd)Equal amount of protein was loaded on 10% SDS-PAGE gel (SDS-PAGE gel preparation kit, AR0138,Boster) and separated.Transfer to PVDF membrane (Millipore, Billerica, MA, USA)The membrane was sealed with 5% skimmed milk (skimmed milk powder, 23210, BD Company, USA) in TBST at room temperature for 1 hour, and mixed with primary antibody alpha-smooth muscle actin (1: 1000, D4K9N, XP, Rabbit mAb, # 19245, CST), vimentin (1: 1000, D21H3, CST) # 5741, CST), phospho-pi3 kinase p85, GAPDH antibody (6c5) loading control (1: 5000, ab8245, abcam) stayed overnight at 4 C .The membrane was washed with TBST and incubated with the second antibody Goat Anti-Rabbit IgG(BA1054, Wuhan boside bioengineering co., ltd.) for 1 hour at room temperature.And then scanned on the exposure machineThen, the comprehensive intensity of each detection band is determined by using image jThese experiments were repeated at least three times.
1.7 Animal experiment
Twelve adult pigmented rabbits were selected, each weighing about 2-2.5kg, After extracted 0.2ml vitreous,12 traumatic PVR model rabbits were prepared by intravitreal injection of 0.1ml platelet rich plasma and 0.1mlPBS[14,15]. They were randomly divided into blank group, control group (0.1mlprp + 0.1mlpbs) and experimental group: (0.1mlPRP + 0.1ml 20ug/ml artesunate). In the control group, 0.1mL platelet-rich plasma and 0.1mL PBS were injected into the vitreous cavity at the same time. In the experimental group, 0.1mL platelet-rich plasma and 0.1ml artesunate (20ug/ml) were injected into the vitreous cavity at the same time. Fundus photography and B-ultrasound machine were used to observe the proliferation of the vitreous and retina at different periods. The expression of Vim, Smad3 and P-Smad3 in vitreous retinal hyperplasia was detected by Western blotting 28 days after surgery, and the retinal tissue structure of each group was observed by frozen section.
1.8 Statistical analysis
All experiments were repeated at least three times with representative results. All experimental data were processed and analyzed using Microsoft Excel and GraphPad Prism 7, and one-way analysis of variance (ANOVA) was used for comparison between groups. Data were expressed as mean ± standard deviation, p < 0.05 was considered statistically significant.