Authors: Wu Di, Sun Xiaonan, Du Lin, Zhang Xiaoyu, Liu Shanshan, Sun Jing, Xu Lin, Zhang Shaodan
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Objective
To investigate the inhibitory effect of rapamycin, an mammalian target of rapamycin (mTOR) pathway inhibitor, on the proliferation, migration and fibrosis of human pterygium fibroblasts (PFBs).
Methods
Pterygium tissues were collected from patients with primary pterygium who underwent surgical excision in Shenyang Fourth People’s Hospital from May to July 2015.The tissues were cultured in vitro and the PFBs were identified by anti-human vimentin immunofluorescence assay.The 3 to 5 generation cells were used for the experiments.The viability of cells treated with different concentrations of rapamycin was detected by methyl thiazolyl tetrazolium (MTT). The cells were divided into normal control group and rapamycin group, and the scratch wound healing test was used to evaluate migration of the PFBs.The expressions of MKI67, α-smooth muscle actin (α-SMA), fibronectin, caspase3, mammalian target of rapamycin (mTOR) and LC3B mRNA were detected by real-time quantitative PCR.
Results
The cultured cells showed morphology of long spindle and were vimentin immunopositive.The cell viability in rapamycin treated PFBs demonstrated a dose-dependent decrease.At 24 hours after culture, The cell viability in 30 μmol/L rapamycin group was (76.67±8.84)% of that in 0 μmol/L rapamycin group (P<0.001). The relative residual scratch width in 30 μmol/L rapamycin group was (35.40±11.62)% 48 hours after scratch, which was significantly greater than (2.45±0.76)% in the normal control group (P<0.05). Real-time quantitative PCR showed that the mRNA expressions of MKI67, α-SMA, fibronectin and mTOR in rapamycin group were significantly decreased when compared with those in normal control group (all at P<0.05). The expression of LC3B mRNA in rapamycin group was significantly higher than that in normal control group (P<0.05). The mRNA expression of caspase3 was not significantly different between the two groups (P=0.861).
Conclusions
Rapamycin can effectively inhibit the proliferation, migration and fibrosis of PFBs without affecting the cell survival.Detailed mechanism remains to be further studied.Rapamycin may serve as an anti-fibrosis agent to prevent the progression and recurrence of pterygium in the future.