| ObjectiveAt present,there are many evidences showed that retinal pigment epithelial(RPE)cells have the potential to trans-differentiate into retinal neuron-like cells through appropriate growth factors activating.And some studies have suggested that the kind of the Traditional Chinese Medicine promoting blood circulation and removing blood stasis can regulate the progress of stem cell differentiation.Hirudo,belonging to the Traditional Chinese Medicine for promoting blood circulation and removing blood stasis,has been confirmed that its own neurons have strong regeneration ability after injuring with complete functional recovery,but we don't know whether Hirudo has the same regulation effect on other stem cell-like cells such as retinal pigment epithelial cells(RPE).Therefore,this study evaluated the effect of Hirudo on the differentiation ability of retinal pigment epithelial cells(RPE).MethodsPart 1 Preliminary study on the effect of Hirudo reprogramming RPE cell.ARPE-19 cells from passages 8-10 were treated with 0.04U/ml Hirudo extract.The morphological changes of RPE cells were observed.CCK-8,EDU and TUNEL were used to determine the effects of Hirudo water extract on the activity,proliferation and apoptosis of RPE cells.To study the effect of Hirudo on the dedifferentiation of RPE cells,real-time PCR was used to quantify the expression of stem cell markers Nestin,Chx10,Pax6,c-Myc and Klf-4 in RPE cells after 72 hours hirudo treatment.To learn the effects of Hirudo on RPE trans-differentiation or directional differentiation into retinal specific cells,the expression of RPE specific markers CRALBP and RPE-65,neurons specific marker TUBB3,retinal ganglion cells specific marker Brn-3b,medium/long wavelength sensitive cone specific marker Opn1 MW / LW,rod specific marker RHO and bipolar specific marker PKC-?,horizontal cell specific marker Calbindin,amacrine specific marker Ch AT,glia specific marker GFAP and fibroblast specific marker Vimentin in RPE cells after 8 days of Hirudo treatment were detected by immunofluorescence staining(IF),real-time PCR and Western blot.Part 2 Promoted the differentiation of RPE cells into retinal ganglion cells(RGC)cells by Hirudo with differentiation medium.ARPE-19 cells were cultured in differentiation medium with B27 and N2 and treated with 0.04U/ml Hirudo extract.The morphological changes of RPE cells were observed.To learn the effects of Hirudo on the trans-differentiation of RPE into retinal ganglion cells and cones,the expression of RPE specific markers CRALBP and RPE-65,neurons specific marker TUBB3 and NF-L,retinal ganglion cells specific marker Brn-3b,Islet1 and Thy-1,medium/long wavelength sensitive cone specific marker OPN1mw/lw were detected by immunofluorescence staining,realtime PCR and Western blot.Part 3 Further promoted the differentiation of RPE cells into retinal ganglion cells(RGC)cells and cones by Hirudo with advanced differentiation medium.ARPE-19 cells were cultured in the differentiation medium with B27,N2,DHA,RA and DAPT for 4 days,and with B27,NGF,IGF-1 and BDNF for another 4 days to promote the progress of RPE differentiation.ARPE-19 cells were treated with 0.04U/ml hirudo extract in the differentiation medium.The morphological changes of RPE cells were observed.To learn the effects of hirudo on the trans-differentiation of RPE into retinal ganglion cells and cones,the expression of RPE specific markers CRALBP and RPE-65,neurons specific marker TUBB3 and NF-L,retinal ganglion cells specific marker Brn-3b,Islet1 and Thy-1,medium/long wavelength sensitive cone specific marker OPN1mw/lw were detected by immunofluorescence staining,realtime PCR and Western blot.ResultsPart 1Treating with Hirudo extract,the body of ARPE-19 cells became smaller,and formed slender processes at the end which were similar to neurons.CCK-8 and EDU showed that Hirudo inhibited the proliferation activity of RPE cells(P < 0.05);TUNEL staining showed that Hirudo had no significant effect on the apoptosis of RPE cells(P > 0.05).Effect of Hirudo on dedifferentiation of RPE cells.After 72 hours treatment of Hirudo,compared with the control group,the expression of stem cell marker KLF-4 in RPE cells increased significantly at the transcriptional level(P < 0.05).And Hirudo had no significant effect on the RPE specific marker CRALBP and other stem cell markers c-Myc,Pax6 and TUBB3 expression in RPE cells(P > 0.05),with Nestin and CHX10 genes were failed to quantified in RPE cells in both groups.After 8 days treatment of Hirudo,the expression of RPE-65(RPE cells)protein decreased(P < 0.05),with Brn-3b(retinal ganglion cells)and OPN1mw/lw(medium/long wave sensitive cone cells)increased significantly,and TUBB3(neuronal cells)decreased in RPE cells compared with the control group(P < 0.05).There was no difference in the expression of RHO(rod cells)and PKC-?(bipolar cell marker),Vimentin(fibroblast)and Pax6(stem cell)in both groups(P > 0.05).At transcription level,the expression of TUBB3(early neuronal marker)in RPE cells increased(P<0.05),the expression of RPE self-specific marker CRALBP and bipolar cell marker PKC-? had no significant changes in RPE cells(P>0.05),and the expression of RPE self-specific marker RPE-65,medium/long wavelength sensitive cone specific marker OPN1mw/lw,rod specific marker RHO,horizontal cell specific marker Calbindin,amacrine specific marker Ch AT and retinal ganglion cells specific marker Brn-3b were failed to quantified in RPE cells in both groups.Part 2With B27 and N2,after 8 days of Hirudo treatment,RPE cells had morphological changes similar to the changes mentioned above,but accompanied by swelling at the end of some processes.The expression of RPE-65 protein in RPE cells of both groups was failed to be detected by Western-blot.The expression of retinal ganglion cells specific marker Brn-3b still increased greatly in RPE cells with Hirudo(P < 0.05),but there was no significant difference in retinal ganglion cells specific marker Islet1 expression(P > 0.05),and retinal ganglion cells specific marker Thy-1 barely expressed in RPE cells of both groups.There were no significant differences in the expression of neurons specific marker TUBB3 and NF-L proteins in RPE cells of both groups(P > 0.05),as well as medium/long wave sensitive cone specific marker OPN1mw/lw protein.Part 3With multiple cytokines,after 8 days of Hirudo treatment,RPE cells had morphological changes similar to the changes mentioned above,and also accompanied by swelling at the end of some processes.The expression of RPE-65 protein in RPE cells of both groups was failed to be detected by Western-blot.The expression of retinal ganglion cells specific marker Brn-3b still increased greatly in RPE cells with Hirudo(P < 0.05),but there was no significant difference in retinal ganglion cells specific marker Islet1 expression(P > 0.05),and retinal ganglion cells specific marker Thy-1 barely expressed in RPE cells of both groups.There were no significant differences in the expression of neurons specific marker TUBB3 and NF-L proteins in RPE cells of both groups(P > 0.05),as well as medium/long wave sensitive cone specific marker OPN1mw/lw protein.ConclusionsHirudo,the Traditional Chinese Medicine,induced morphological changes of RPE cells similar to neurons.Down-regulated the expression of RPE specific marker RPE-65 protein,and up-regulated the expression of Brn-3b protein,a specific marker of early retinal ganglion cells.Hirudo also up-regulated the expression of OPN1 mw/lw,a marker of medium and long wave sensitive cone cells,without differentiation medium.And there was no significant effect of Hirudo on the expression of other retinal cell markers.All these results suggested that Hirudo might induce directional differentiation of RPE cells into special kind of retinal neurons,especially early retinal ganglion progenitor cells.Our results provide a new experimental basis for further understanding the effect of Hirudo on cell differentiation and regeneration,and a new insight for its application in retinal degenerative diseases. |
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