RESUMO
Background Our previous study demonstrated that the aptamer S58 specifically targeted transforming growth factor-β receptor Ⅱ (TβRⅡ) and inhibited the transdifferentiation of human Tenon capsule fibroblasts (HTFs) mediated by transforming growth factor-β (TGF-β).Chitosan-nanoparticles (CS-NP) are good drug carriers,but the efficacy and safety of CS-NP/aptamer complexes deserve attention.Objective The aim of this study was to synthesize a novel CS-NP/aptamer complex called CS (S58)-NP and investigate its properties and applicability.Methods Human Tenon capsule tissue was obtained from patients during strabismus surgery,and HTFs were cultured and passaged using the explant culture method.The fourth to tenth generations of cells were used in the experiment.Different concentrations of CS-NP were used to prepare the CS(S58)-NP by the ionic cross-linking method with a surface charge rate (N/P) for S58 of 10,20,30 or 40.The particle size and Zeta potential were measured by the Zeta analyzer.The shape and distribution of CS (S58)-NP particles were examined under the scanning electron microscope.The binding of CS-NP with S58 and resistance of CS (S58)-NP to DNase Ⅰ were examined by agarose gel eletrophoresis.The release rate of S58 from CS (S58)-NP in PBS was quantitatively analyzed by a ultraviolet spectrophotometer.The cytotoxicity of CS(S58)-NP to HTFs was evaluated by detecting the production of lactate dehydrogenase (LDH).Results The Zeta analyzer showed that the particle size of CS (S58)-NP was 130-270 nm and its electric potential ranged from + 16 to +28 mV.The CS (S58)-NP particles appeared spherical with an even distribution under the scanning electron microscope.The mean encapsulation efficiency of CS(S58)-NP was 88.9％,89.3％,91.7％ or 90.5％,respectively,when the N/P was 10,20,30 or 40.After being encapsuled by CS-NP,S58 could resist the degradation from DNase I.Its total releasing level in PBS increased with the lapse of time,with a maximum releasing speed at 24 to 36 hours.The total releasing level reached 100％ at 96 hours.With increaseing concentrations of CS(S58)-NP,the relative releasing level of LDH in HTFs suspension gradually elevated with a significant difference among the groups (F =588.018,P =0.000),with the highest released LDH level at 50 nmol/L of CS(S58)-NP (12.853％ ±0.375％).Conclusions CS-NP provides a protective and slow-releasing effect on the S58 aptamer.CS (S58)-NP shows a good biocompatibility with HTFs with a low cytotoxicity at a concentration of ＜50 nmol/L.CS(S58)-NP could be used to inhibit TGF-β induced transdifferentiation of HTFs in the future.
RESUMO
Background Research showed that transforming growth factor-β2 (TGF-β2) promotes scar formation.But its mechanism in scarring after glaucoma filtration surgery is worthy of studying.Objective This study was to investigate the effect of TGF-β2 on myofibroblast transition of human Tenon fibroblasts (HTFs) and scarring after glaucoma filtration surgery.Methods Tenon capsular tissue was obtained from 3 patients with strabismus during the surgery and was incubated in DMEM with 10％ fetal bovine serum (FBS).The cells were collected and passaged in the free-serum medium for 24 hours,and then 1,2,5,10,20 μg/L TGF-β2 was added into the medium respectively,to induce the transformation of HTFs,and 2 μg/L or 5 μg/L TGF-β2 was used to treat the HTFs for 6,24,48 and 72 hours.The control group was not treated with TGF-β2.The expressions of α-smooth muscle actin (α-SMA) and phosphorylation of the signaling proteins (pSmad2) in HTFs were detected by Western blot assay.The expressions of α-SMA and F-actin were located by cell immunofluorescine technique under the confocal immunofluorescence microscopy.Cell contractility was determined by collagen gel contraction assays.This study was approved by Ethic Committee of Institute of Surgery Research of Daping Hospital,and informed consent was obtained from each patient or custodian initial of the study.Results The expression of α-SMA protein in the HTFs was increased significantly after the treatment of TGF-β2 in comparison with the control group and reached a peak at 24-48 hours.The α-SMA expression was gradually weakened in the 10 μg/L TGF-β2 groups.Little of α-SMA and F-actin were expressed in the control group.However,strong staining for α-SMA and F-actin were observed in the 1,2 and 5 μg/L TGF-β2 groups and then the staining weakened at the concentration of 10 μg/L.In addition,pSmad2 showed a stronger expression in the 2 μg/L TGF-β2 group than that in the PBS group and FBS group,with the strongest expression in 30 minutes through 2 hours.The untreated gel contracted (78.00±3.13)％ from its initial size,and contraction in the 1,2,5,10 μg/L TGF-β2 group were (63.88±1.78)％,(20.69±0.65)％,(19.49-±0.54)％,(16.24±0.84) ％,respectively,TGF-β2 increased HTFs contraction significantly (Fgroup =859.400,P =0.000).Conclusions TGF-β2 can induce transdifferentiation of Tenon fibroblast into myofibroblast and increase cell contractility,with a concentration-dependent and time-dependent pattern to an extent.It may be the mechanism of scar formation after glaucoma filter surgery.