Pressure-induced differentiation of rabbit retinal stem cells into retinal ganglion cells
10.3760/cma.j.cn115989-20200519-00361
- VernacularTitle:压力诱导兔视网膜干细胞向视网膜神经节细胞分化研究
- Author:
Min DAI
1
;
Zefeng KANG
;
Zhulin HU
;
Yan LI
Author Information
1. 云南大学附属医院眼科 云南省眼科医院 云南省眼科研究所 云南省眼科疾病防治研究重点实验室 云南省第二人民医院白内障与眼底疾病防治省创新团队 云南省姚克专家工作站 云南省眼部疾病临床医学研究中心 云南省眼科疾病临床医学中心，昆明 650021
- Keywords:
Retina;
Stem cells;
Retinal ganglion cells;
Pressure;
Cell differentiation;
Glaucoma
- From:
Chinese Journal of Experimental Ophthalmology
2022;40(12):1134-1140
- CountryChina
- Language:Chinese
-
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.
