ABSTRACT
Objective:To investigate the effect of pressure on the differentiation of rabbit retinal stem cells (RSCs) co-cultured with retinal ganglion cells (RGCs).Methods:SPF grade New Zealand rabbits on the day 22 of gestation were selected, and embryos were removed to obtain retinal ciliary margin pigment epithelial tissue and culture primary RSCs.Six SPF grade newborn New Zealand rabbits were selected, and retinal neuroepithelial layer tissues were isolated to culture primary RGCs.Rabbit RSCs cultured in vitro were identified by immunofluorescence staining of nestin antibody, bromodeoxyuridine (BrdU) cell proliferation assay kit, RSCs spontaneously differentiated cells immunofluorescence detection and flow cytometry.RGCs were identified through immunofluorescence staining of Brn3b antibody and Thy1.1 antibody.A co-culture system of RGCs and RSCs cultured in the upper and lower layers of a transwelll plate respectively was constructed.The mRNA and protein expression levels of nestin and Thy1.1 in RSCs and differentiated cells under pressures of 0, 20, 40, 60, 80 mmHg (1 mmHg=0.133 kPa) were detected by real-time fluorescence quantitative PCR and Western blot.The feeding and use of laboratory animals were in accordance with the Regulations on the Administration of Laboratory Animals promulgated by the State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Yunnan University Affiliated Hospital (No.KPRC-IACUC17008). Results:RSCs cultured in vitro were nestin-positive.The percentage of BrdU-positive isolated RSCs was (92.26±3.28)%.Some cells differentiated from RSCs were Brn3b-positive, accounting for (13.00±3.06)%, and some were GS-positive, accounting for (31.60±3.67)%.RGCs cultured in vitro were Brn3b- and Thy1.1-positive.There were statistically significant differences in the relative mRNA and protein expressions of nestin and Thy1.1 between RSCs and differentiated cells under different pressures (mRNA: F=127.600, 137.400; both at P<0.01; protein: F=82.480, 158.700; both at P<0.001). The relative mRNA and protein expressions of nestin were significantly reduced in RSCs, and relative mRNA and protein expressions of Thy1.1 were significantly increased in differentiated cells at 20, 40, 60 and 80 mmHg in comparison with 0 mmHg (all at P<0.05). When the pressure was 40 mmHg, the relative mRNA and protein expressions of nestin were lowest in RSCs, and the relative mRNA and protein expressions of Thy1.1 in differentiated cells were highest. Conclusions:Within a certain range, pressure can promote the differentiation of RSCs co-cultured with RGCs into ganglion-like cells, and excessive pressure can inhibit the differentiation of RSCs.
ABSTRACT
Objective:To study the mechanism of the effect of Jiajian-Zhujing Decoction on the expression of VEGF on ARPE-19 cells after AKT transfection. Methods:To prepare the serum and blank serum of Jiajian-Zhujing Decoction and divide ARPE-19 cells into the normal group, model group, blank serum group, medicated serum group, Conbercept group and combined group. Except normal group, this research established AKT transfected cell model. Then cultured the normal group and model group with conventional method, and the blank serum group was cultured with 10% blank serum, the medicated serum group was cultured with 10% medicated serum, the Conbercept group was cultured with 20 μg/ml Conbercept, the combined group was cultured with 10% medicated serum and 20 μg/m Conbercept. The proliferation of ARPE-19 cells in each group was detect by the CCK-8 method. The levels of AKT, mTOR and VEGF mRNA were detected by real-time quantitative PCR. Western blot was used to detect the expression of AKT, mTOR and VEGF. Results:After being cultured for 24, 48 and 72 hours, compared with the model group, the cell proliferation rate in blank serum group, medicated serum group, Conbercept group and combined group significantly decreased ( P<0.05). Compared with the model group, the expression of AKT mRNA (24 h: 3.10 ± 0.48, 1.97 ± 0.14, 1.26 ± 0.24 vs. 4.77 ± 0.68; 48 h: 3.52 ± 0.82, 2.62 ± 0.77, 1.10 ± 0.19 vs. 6.12 ± 1.21), mTOR mRNA (24 h: 3.02 ± 0.26, 2.45 ± 0.75, 1.13 ± 0.15 vs. 4.48 ± 0.80; 48 h: 1.29 ± 0.30, 1.30 ± 0.57, 0.65 ± 0.19 vs. 2.54 ± 0.62), VEGF mRNA (24 h: 3.33 ± 0.62, 2.18 ± 0.20, 1.55 ± 0.28 vs. 5.53 ± 1.02; 48 h: 2.35 ± 0.54, 1.23 ± 0.28, 0.93 ± 0.25 vs. 3.59 ± 0.40), AKT protion (24 h: 0.45 ± 0.09, 0.25 ± 0.05, 0.14 ± 0.04 vs. 0.62 ± 0.04; 48 h: 0.36 ± 0.06, 0.23 ± 0.04, 0.14 ± 0.03 vs. 0.54 ± 0.08), mTOR protion (24 h: 0.35 ± 0.05, 0.24 ± 0.02, 0.18 ± 0.02 vs. 0.52 ± 0.09; 48 h: 0.23 ± 0.04, 0.29 ± 0.04, 0.14 ± 0.03 vs. 0.40 ± 0.10), VEGF protion (24 h: 0.14 ± 0.03, 0.33 ± 0.04, 0.24 ± 0.03 vs. 0.54 ± 0.10; 48 h: 0.24 ± 0.03, 0.17 ± 0.02, 0.11 ± 0.02 vs. 0.42 ± 0.10) significantly decreased ( P<0.05), and the combined group was significantly lower than that of the Conbercept group ( P<0.05). Conclusions:AKT transfection can promote the proliferation of ARPE-19 cells, and Jiajian-Zhujing Decoction can significantly inhibit this proliferation. Jiajian-Zhujing Decoction may inhibit the activity of AKT/mTOR signaling pathway to reduce the expression of VEGF.
ABSTRACT
Background Choroidal neovascularization (CNV) of macular zone is one of blinding eye diseases.Conventional treatment methods include photodynamic therapy and intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs.However,there are some adverse effects following these treatments.Researches showed that fiajianzhujing decoction,a Chinese herbal medicine,can inhibit CNV.Objective This study was to investigate the impact of drug serum offiajianzhujing decocation (drug serum) on the proliferation of rat choroidal vascular endothelial cells (RCVECs).Methods Jiajianzhujing decoction (0.525 g/ml) was used by intragastric administration with the dosage 20 ml/kg for 3 days,and the abdominal aortic blood was collected 2 hours after last dosage to prepare the drug serum.Cultured RCVECs were divided into 4 groups.CoCl2 with the concentration of 100 μmol/L (100 μl) was added in the medium to establish the anoxic models in the CoCl2 group,and 20％ drug serum was added in the anoxic culture medium in the drug serum+CoCl2 group.Regular culture medium was used in the blank control group,and 20％ pure serum was added in the anoxic culture medium in the pure serum+CoCl2 group.The cells were incubated for consecutive 24 hours,and the growth of the cells was detected by monotetrazolium (MTT) assay as absorbance (A).The expressions of gene and protein of hypoxia-inducible factor-1 α (HIF-1 α) and VEGF in the cells were detected by reverse transcription PCR and Western blot,respectively.This study was approved by the Ethics Committee of Eye Hospital of China Academy of Chinese Medical Sciences.Results Cultured cells grew well with fusiform shape.Hypoxic cell models were created by adding CoCl2.The mean A values were 0.659± 0.051,0.757±0.553,0.683±0.037 and O.731 ±O.038 in the blank control group,CoCl2 group,drug serum+CoCl2 group and pure serum+CoCl2 group,respectively,and the A value in the CoCl2 group was significantly elevated in comparison with the blank control group and drug serum+CoCl2 group (both at P＜0.O1).The significant differences were found in the relative expressions of HIF-1 α and VEGF among the blank control group,CoCl2 group,drug serum+ CoC12 group and pure serum+CoCl2 group (HIF-1 α:F =3.100,P＜0.05;VEGF mRNA:F =3.420,P＜0.05;HIF-1 α protein:F=470.600,P =0.000;VEGF protein:F =146.700,P =0.000),and the relative expressions of HIF-1α mRNA,VEGF mRNA,HIF-1α protein and VEGF protein in the CoCl2 group were significantly higher than those in the blank control group and drug serum+CoCl2 group (all at P＜0.05).Conclusions Drug serum ofjiajianzhujing decoction can inhibit the growth and proliferation of hypoxic RCVECs by down-regulating the secretion of HIF-1α and VEGF.