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Jing Zhu, Xinyi Wu, LIQUN DU; SPARC Promotes Self-Renewal of Limbal Stem Cell through the phosphorylation of p38-MAPK signal pathway. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3873. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the function of secreted protein acidic and rich in cysteine (SPARC) in the maintenance of limbal stem cells (LSCs) physiological function.
LSCs were treated with different concentration of exogenous SPARC (5μg/ml, 1μg/ml, 0.5μg/ml, 0.1μg/ml and 0μg/ml), the marker expression (p63,ABCG-2,Bmi-1,CK3/12), mitogenic effect and holoclone forming capacity of cultured LSCs were analyzed by immunofluorescent staining, PCR, CCK-8 test, EdU labeling and colony-forming efficiency test. To explore the mechanism of SPARC in the stemness preservation of LSCs, the phosphorylation of signal pathway p38-MAPK,ERK, JNK, STAT3 and PI3K was evaluated by western blot and immunofluorescent staining.
Immunofluorescent staining showed that exogenous SPACR increased the expression of LSCs specific markers such as p63,ABCG-2,Bmi-1 and decreased the expression of differentiation marker CK3/12 in a concentration - dependent manner. EdU assay demonstrated that SPARC stimulated LSCs proliferation,in the group of 5μg/ml and 1μg/ml over 12% p63-positive cells showed EdU-positive, and in 0μg/ml group only less than 4% p63-positive cells were labeled by EdU. Due to exogenous SPARC inhibited the adhesion of differentiated corneal epithelial cells, CCK-8 and CFE test showed a slight decline in 5μg/ml and 1μg/ml group, however the clone morphology in these 2 group was more uniform and regular. With 1μg/ml SPARC, the activation of phosphorylated p38-MAPK was observed by western blot and immunofluorescent staining. Signal pathway inhibitor of p38-MAPK further validated this result.
SPARC mediate the stemness preservation of LSCs through the phosphorylation of p38-MAPK signal pathway.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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