| Objective: 1.To observe the effects of different concentrations of high-sugar culture on human retinal pigment epithelial(h RPE)cells.2.Observe the expression changes of small nucleolar RNA host gene 1(SNHG1)in h RPE cells induced by high glucose,and its role in the Epithelial mesenchymal transformation(EMT)and inflammation of h RPE cells induced by high glucose.Methods: 1.Observe the effects of different concentrations of high glucose culture on h RPE cells.ARPE-19 cells were divided into(1)normal group: 5.5m M glucose cultured for 48h;(2)30m M hypertonic group: 30.0m M hypertonic medium cultured for 48h;(3)30m M high glucose group: 30.0m M glucose cultured for 48h;(4)60m M hypertonic group: 60.0m M hypertonic medium cultured for 48 h.(5)60m M high glucose group: 60.0m M glucose cultured for 48h;RT-PCR detection of epithelial markers E-cadhrein,tight junction protein(ZO-1)expression level and Vimentin and α-SMA expression levels;CCK8 assay was used to detect the proliferation activities of ARPE-19 cells in each group.2.The expression of SNHG1 in h RPE cells cultured in high glucose.60m M high glucose stimulated ARPE-19 cells for 0h,12 h,24h,48 h,72h,and RT-PCR was used to detect the expression changes of SNHG1.3.The effect of SNHG1 on EMT and inflammation of h RPE cells induced by high glucose ARPE-19 cells are divided into(1)normal control group: 5.5m M glucose cultured for 48h;(2)hypertonic control group: osmotic pressure 60.0m M hypertonic medium cultured for 48h;(3)high glucose intervention group: 60.0m M high glucose Cultured for 48h;(4)si-NC group:After transfection of si-con SNHG1 into cells,cultured with 60.0m M high glucose for 48h;(5)si SNHG1 group: After transfected si-SNHG1 into cells,cultured with 60.0m M high glucose for 48 h.RT-PCR detects the expression of SNHG1 and the expression of E-cadhrein,ZO-1,Vimentin,and α-SMA in each group of cells;CCK-8 assay detects the proliferation activity of each group of cells;cell scratch test detects the migration area of each group of cells;Annexin V-FITC/PI double-labeled flow cytometry was used to detect the apoptotic rate of each group of cells;ELASA method was used to detect the protein expression of IL-6 and IL-1β in each group of cells.Results:1.The effect of different concentrations of glucose culture on the EMT of h RPE cells.RT-PCR results showed that the expressions of E-cadhrein and ZO-1 in the 30 m M high glucose group and 60 m M high glucose group were significantly lower than the normal group and the expressions of Vimentin and α-SMA were significantly higher than the normal group(P<0.05).The expression of E-cadhrein and ZO-1 in the 60 m M high glucose group was lower than that of the 30 m M high glucose group,and the expression of Vimentin and α-SMA was higher than that of the 30 m M high glucose group(P<0.05).The results of CCK8 experiment showed that the cell proliferation ability of the 30 m M high glucose group was not significantly different from that of the normal group(P>0.05),and the cell proliferation ability of the 60 m M high glucose group was significantly higher than that of the normal group and the 30 m M high glucose group(P<0.05).2.The expression of SNHG1 in h RPE cells cultured with high glucoseSNHG1 increased in a time-dependent manner under 60 m M high-sugar culture conditions,and reached the highest at 48 h.3.The effect of SNHG1 on EMT and inflammation of h RPE cells induced by high glucose RT-PCR results showed that the expressions of E-cadhrein and ZO-1 in the cells of the high glucose intervention group were lower than those of the control group,and the expressions of Vimentin and α-SMA were higher than those of the control group(P<0.05);Si-SNHG1 group E-cadhrein and ZO-1 were higher than si-NC group,Vimentin and α-SMA were lower than si-NC group(P<0.05).The results of the cell scratch experiment showed that the 48 h migration area of the high glucose intervention group was significantly larger than that of the control group(P < 0.05),and the 48 h migration area of the si-SNHG1 group was significantly smaller than that of the Si-NC group(P < 0.05).The results of the CCK-8experiment showed that the cells in the high glucose intervention group had higher proliferation activity than the control group(P<0.05),and the cells in the Si-SNHG1 group had lower proliferation activity than the Si-NC group(P < 0.05).The results of flow cytometry showed that the apoptosis rate of the high glucose intervention group was significantly lower than that of the control group(P<0.05),and the apoptosis rate of the si-SNHG1 group was significantly higher than that of the Si-NC group(P<0.05).ELASA results showed that the levels of IL-6 and IL-1β in the cells of the high glucose intervention group were higher than those in the control group(P<0.05).The levels of IL-6 and IL-1β in the si-SNHG1 group were lower than those in the si-NC group(P<0.05).Conclusion: 1.High glucose culture can induce epithelial-mesenchymal transition of h RPE cells.2.The expression of SNHG1 increased in a time-dependent manner in h RPE cells cultured with high glucose,and it participated in the epithelial-mesenchymal transition and inflammation process of h RPE cells.3.SNHG1 promoted the proliferation and migration of h RPE cells and inhibited their apoptosis by promoting epithelial-mesenchymal transition. |
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