Experimental Study On The Expression Of SLC39A5 In Human Fetal Scleral Fibroblasts And Its Relationship With TGF-?2?MMP-2

Objective To investigate the expression of SLC39A5 in human fetal sclera fibroblasts(HFSFs).And the effect of myopia factor TGF-?2 on its expression.To observe the expression of matrix metalloproteinase 2(MMP-2)after intervention(up-regulation / down-regulation)the expression of SLC39A5.To analyze the relationship between SLC39A5 and TGF-?2 signaling pathway.To explore the role of SLC39A5 in sclera remodeling and myopia pathogenesis.Methods 1.The HFSFs were subcultured.The 5-7 generations of well-formed HFSFs were used for the experiment.2.The expression of SLC39A5 in normal human fetal scleral fibroblasts was detected by QRT-PCR,Western-blot and immunofluorescence.The THP-1 cells were positive control.To determine the expression and localization of m RNA and protein of SLC39A5 in HFSFs.3.Stimulated the HFSFs with TGF-?2 of different concentration(2.5?mol / L,5?mol / L,10?mol / L,20?mol / L),the stimulate time was 4h,8h,16 h respectively.Detected the expression of MMP-2 and SLC39A5 by QRT-PCR and Western-blot.4.After the expression of SLC39A5 was down-regulated and up-regulated by RNA interference and recombinant protein,the expression of SLC39A5?MMP-2 in HFSFs were detected to observe the change of MMP-2.Results 1.The HFSFs were successfully subcultured.2.The expression of SLC39A5 in normal HFSFs was detected by QRT-PCR and immunofluorescence.When the stimulatory concentration of TGF-?2 increased gradually from 2.5?mol / L to 20?mol / L,stimulation time increased from 4h to 16 h,the expression of MMP-2 m RNA in HFSFs was significantly increased.And the expression of MMP-2 m RNA reached the peak at the concentration of 5?mol / L and the time of 8h.And the expression of SLC39A5 m RNA was increase at this time comparing with the normal control.The differences were statistically significant(P <0.05).The expression of MMP-2 also increased after the HFSFs were stimulated with TGF-?2,confirming that TGF-?2 had an up-regulation effect on MMP-2 in HFSFs.4.Down-regulated / up-regulated SLC39A5 of the HFSFs at the peak time by RNA interference and recombinant proteins respectively.Western-blot showed that the expression of SLC39A5 protein changed afterward,the difference was statistically significant(P <0.05).While the expression of MMP-2 did not change significantly.Conclusions 1.The m RNA and protein of SLC39A5 were expressed in HFSFs.2.The expression of SLC39A5 in HFSFs was significant increase after stimulated with TGF-?2.Suggest that SLC39A5 may be involved in the formation of myopia with TGF-?2.3.The expression of MMP-2 in HFSFs did not change significantly after downregulated and upregulated the expression of SLC39A5,suggesting that mechanism of SLC39A5 involved in myopia may be independent with MMP-2.