| Study background and purpose:Keratopathy is a common blinding eye disease in the world,which is second only to cataract.At present,there are still more than 10 million patients with corneal blindness.Corneal transplantation has been the most effective solution to many corneal diseases.But in recent years,In recent years,there is a growing shortage of corneal materials that can be donated and used in clinical practice.So it is difficult to meet the needs of patients.About 38% of 185000 corneas transplanted every year in the world are related to endothelial dysfunction.The density of corneal endothelial cells decreased as the increasing of age.The average annual decrease of corneal endothelial cells in healthy adults was about 0.6% ± 0.5%.When the number of endothelial cells decreased to a certain extent,the function of corneal endothelial barrier was destroyed,and the endothelial function was decompensated,leading to corneal edema and bullous keratopathy.Compared with penetrating keratoplasty,corneal endothelial transplantation has the advantages of shorter recovery time,better postoperative vision and less immune rejection.At present,it has gradually replaced penetrating keratoplasty and is widely used in the treatment of corneal endothelial dysfunction caused by various reasons.Due to the limited number of donor corneas,the development of various artificial biomaterials can alleviate the shortage of human donor corneas to a certain extent.A number of studies on these alternative corneal materials have been reported.In recent years,the culture of human corneal endothelial cells(HCECs)in vitro has become a research hotspot.The transplantation of cultured HCECs may be a future treatment alternative to corneal endothelial transplantation.Once this method is used in clinic,it can alleviate the serious shortage of corneal donor.The main problem of HCECs culture in vitro is how to enhance the proliferation of HCECs and maintain its functional characteristics.At present,it is still a hot and difficult point to find a method to promote the proliferation of HCECs.The aim of this study was to explore the potential role of Human amniotic epithelial cells(HAECs)in the proliferation of human corneal endothelial cells(HCECs)and the possible mechanism of regulation.Methods:1.The human amniotic membrane tissue and human corneal tissue were collected separately,and then both of the primary cells of HAECs and HCECs were isolated and cultured,and the identification was performed by flow cytometry;2.In vitro,after HAECs were cultured and then the supernatant of the medium was collected to prepare the HAECs-CM culture system;The HCECs were divided into four groups,including the corneal endothelium medium group(CEM),the 20%human amniotic epithelial cell culture medium group(20% HAEC-Me),the 20%human amniotic epithelial cell-conditioned medium group(20% HAEC-CM),and the 20% human amniotic epithelial cell-conditioned medium supplemented with GSk-3β inhibitor group(GSk-3β inhibitor).3.CCK-8 assay was used to detect the proliferation capacity of HCECs,and flow cytometry was used to detect the apoptosis and cell cycle changes of HCECs;4.Flow cytometry was used to detect the changes of ROS and mitochondrial membrane potential;5.Immunofluorescence assay was used to detect the α-SMA and β-catenin expression;6.The telomere length and telomerase activity of HCECs were detected by PCR and ELISA;7.The Wnt/β-catenin pathway related genes and protein expression were measured by RT-PCR and Western blot;Results:1.Flow cytometry results showed that the positive rate of keratin 19(CK19)of HAECs was 99%,the negative rate of Vimentin was more than 98%,and the positive rate of NSE of HCECs was 97%,and the negative rate of Vimentin was more than 97%,indicating we successful isolated the HAECs and HCECs;2.The 20% HAEC-CM culture system can promote the proliferation of HCECs,inhibit the apoptosis of HCECs,and significantly reduce the proportion of G1cells;3.The 20% HAEC-CM culture system can reduce the ROS level and decrease the cell proportion of mitochondrial membrane potential decline of HCECs;4.The 20% HAEC-CM culture system can reduce the expression of α-SMA and promote the expression of β-catenin and translocates to the nucleus;5.20% HAEC-CM culture system can increase telomere length and telomerase activity of HCECs;6.20% HAEC-CM culture system can promote the expression of transcription factor 4(Tcf4)and activate the Wnt /β-catenin pathway;7.After activating Wnt/β-catenin pathway,the effects of 20% HAEC-CM culture system on HCECs proliferation and telomerase activity is enhanced.Conclusion:The microenvironment of human amniotic epithelial cells can promote the proliferation of human corneal endothelial cells and reduce the oxidative damage of human corneal endothelial cells,which may be related to the regulation of telomerase activity by the Wnt /β-catenin pathway. |
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