Effect Of Regulating The Expression Of HSP47 On Collagen Metabolism Of Scleral Fibroblasts

Objective: To explore the effect of HSP47 on synthesis,secretion and degradation of collagen in scleral fibroblasts by regulating the gene expression of HSP47.Methods: Scleral fibroblasts were cultured in vitro from 4 weeks old normal guinea pigs,3 to 5 generations were used for the experiments.The eukaryotic vector of heat shock protein 47(HSP47)gene constructed by molecular cloning was transfected into fibroblasts.HSP47 siRNA was synthesized by chemical method and transfected into fibroblasts.The mRNA and protein expression of HSP47,collagen I,collagen III,collagen V and alpha-smooth muscle actin(α-SMA)were detected by quantitative real-time polymerase chain reaction(qRT-PCR)and western blot(WB).The protein expressions of matrix metalloproteinase 2(MMP-2),tissue inhibitor of matrix metalloproteinase1/2(TIMP-1/2),collagen I,collagen III and collagen V in the extracellular matrix were detected by enzyme-linked immunosorbent assay(ELISA).The internal structure of fibroblasts was observed by transmission electron microscopy.Results: There was no significant difference between the normal control group and the negative control group(P > 0.05).qRT-PCR: Compared with the normal control group,the mRNA expressions of HSP47,collagen I and α-SMA in the HSP47 up-regulated group were increased(all P < 0.05),while the mRNA expressions of collagen III and V were not significantly changed(all P > 0.05).The mRNA expression of HSP47 and type V collagen in the HSP47 down-regulated group was decreased(all P < 0.05),and the mRNA expression of type I,III collagen and α-SMA was not significantly changed(all P >0.05).Compared with the negative control group,the mRNA expression of HSP47 and collagen I in the HSP47 up-regulated group increased(all P < 0.05),while the mRNA expression of collagen III,V and α-SMA showed no significant changes(all P >0.05).The mRNA expression of type III and type V collagen was decreased in the HSP47down-regulated group(all P < 0.05),while the mRNA expression of HSP47,type I collagen and α-SMA was not significantly changed(all P > 0.05).WB: Compared with the normal control group,the protein expressions of HSP47,type I,type III collagen andα-SMA in HSP47 up-regulated group were increased(all P < 0.05),while the protein expression of type V collagen was not significantly changed.In the HSP47 down-regulated group,the expression of HSP47,type I and type III collagen was reduced(all P < 0.05),while the expression of V collagen and α-SMA was not significantly changed(all P >0.05).Compared with the negative control group,the protein expressions of HSP47,type I,type III,type V collagen and α-SMA in HSP47 up-regulated group were significantly increased(all P < 0.05).In the HSP47 down-regulated group,the protein expressions of HSP47 and collagen I and V were reduced(all P < 0.05),while the protein expressions of collagen III and α-SMA were not significantly changed(all P > 0.05).ELISA: Compared with the normal control group,the protein expression of type I,III,V collagen,TIMP-1 and TIMP-2 in the HSP47 up-regulated group increased(all P < 0.05),while the protein expression of MMP-2 decreased(P < 0.05).And the down-regulated group did the opposite.Compared with the negative control group,it is almost the same.Conclusion:(1)Upregulation of HSP47 can promote the synthesis and secretion of type I,III and V collagen by scleral fibroblasts,and delay the degradation of collagen.Down-regulation of HSP47 can inhibit the synthesis and secretion of type I,III and V collagen by scleral fibroblasts,and promote the degradation of collagen.(2)Upregulation of HSP47 can promote the expression of collagen I mRNA,while downregulation of HSP47 has no inhibitory effect on the expression of collagen I mRNA.(3)HSP47 can promote the transdifferentiation of scleral fibroblasts into myofibroblasts.