AIM：To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix（APCM） in vitro.·METHODS：The scaffold w...AIM：To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix（APCM） in vitro.·METHODS：The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate（SDS）solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin（HE）staining and 4’,6-diamidino-2-phenylindole（DAPI）staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·R ESULTS：Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION：Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vitro.展开更多
AIM: To explore the protective effect of zeaxanthin on human limbal and conjunctival epithelial cells against UVradiation and excessive oxidative stress.METHODS: Human limbal and conjunctival epithelial cells were iso...AIM: To explore the protective effect of zeaxanthin on human limbal and conjunctival epithelial cells against UVradiation and excessive oxidative stress.METHODS: Human limbal and conjunctival epithelial cells were isolated from cadaver and cultured in vitro. They were challenged with UVB radiation and H2 O2 with and without zeaxanthin pretreatment. Cell viability, p38 and c-JUN NH(2)-terminal kinase(JNK) phosphorylation, IL-6, IL-8 and MCP-1 secretion and malondialdehyde(MDA) content were measured.RESULTS: Zeaxanthin had no measurable cytotoxicity on limbal or conjunctival epithelial cells when used at concentrations of 5 μg/mL and below. At 30 mJ/cm2 UVB, the pretreatment of zeaxanthin increased the percentage of live cells from 50% to 69%(P=0.01) and from 66% to 75%(P=0.05) for limbal and conjunctival epithelial cells, respectively. The concentrations of IL-6, IL-8 and MCP-1 in the culture medium reduced to 66%(for IL-6 and MCP-1)and 56%(for IL-8) of the levels without zeaxanthin. This was accompanied by reduced p38 and JNK protein phosphorylation. Pretreatment of zeaxanthin also reduced intracellular MDA content caused by H2 O2 stimulation from 0.86 μmol/L to 0.52 μmol/L(P=0.02) in limbal epithelial cells and from 0.96 μmol/L to 0.56 μmol/L in conjunctival epithelial cells(P=0.03). However, zeaxanthin did nothave significant effect on H2 O2-induced cell death in limbal or conjunctival epithelial cells.CONCLUSION: Zeaxanthin is an effective reagent in reducing the detrimental effect of UV-radiation and oxidative stress on ocular surface epithelial cells.展开更多
AIM: To determine the proliferative potential and the maintenance of stem cell activity in stored human limbal tissues, and correlate this with the preservation time, cell viability and the expression of stem cell mar...AIM: To determine the proliferative potential and the maintenance of stem cell activity in stored human limbal tissues, and correlate this with the preservation time, cell viability and the expression of stem cell markers. METHODS: Thirty limbal rims were split into 4 parts and stored in corneal preservation medium at 4 degrees C for 0, 1, 4, or 7 days. The limbal stem cell and mitotic markers P63, CK19, proliferating cell nuclear antigen (PCNA), and Ki67 were determined by immunohistochemical staining. The proliferative potential of limbal epithelial cells was assessed by cell viability, the ability of generating stratified epithelium, and colony forming assay. RESULTS: The stored tissues maintained limbal stratified structure to 7 days and exhibited comparable expression level of stem cell and mitotic markers. The proportion of viable cells decreased with the prolonged preservation time, while colony forming efficiency decreased from the 1st day and disappeared at the 4th day. When inoculated on amniotic membrane, the cells preserved for 1 day formed a stratified epithelium, while the cells from 4 days' preservation formed a discontinuous layer. CONCLUSION: The colony forming efficiency of limbal epithelial stem/progenitor cells decreased rapidly with the increasing preservation time, while the expression level of markers and capacity of forming epithelial monolayer on amniotic membrane decreased gradually. The limbal epithelial stem cells lost their function earlier than the lost expression level of stem cell markers. This may help us to better choose the appropriate preservation grafts for future limbal stem cell transplantation.展开更多
基金Supported by the National Natural Science Foundation of China(No.81271716)
文摘AIM：To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix（APCM） in vitro.·METHODS：The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate（SDS）solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin（HE）staining and 4’,6-diamidino-2-phenylindole（DAPI）staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·R ESULTS：Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION：Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vitro.
基金Supported by the National Natural Science Foundation of China(No.81570829)
文摘AIM: To explore the protective effect of zeaxanthin on human limbal and conjunctival epithelial cells against UVradiation and excessive oxidative stress.METHODS: Human limbal and conjunctival epithelial cells were isolated from cadaver and cultured in vitro. They were challenged with UVB radiation and H2 O2 with and without zeaxanthin pretreatment. Cell viability, p38 and c-JUN NH(2)-terminal kinase(JNK) phosphorylation, IL-6, IL-8 and MCP-1 secretion and malondialdehyde(MDA) content were measured.RESULTS: Zeaxanthin had no measurable cytotoxicity on limbal or conjunctival epithelial cells when used at concentrations of 5 μg/mL and below. At 30 mJ/cm2 UVB, the pretreatment of zeaxanthin increased the percentage of live cells from 50% to 69%(P=0.01) and from 66% to 75%(P=0.05) for limbal and conjunctival epithelial cells, respectively. The concentrations of IL-6, IL-8 and MCP-1 in the culture medium reduced to 66%(for IL-6 and MCP-1)and 56%(for IL-8) of the levels without zeaxanthin. This was accompanied by reduced p38 and JNK protein phosphorylation. Pretreatment of zeaxanthin also reduced intracellular MDA content caused by H2 O2 stimulation from 0.86 μmol/L to 0.52 μmol/L(P=0.02) in limbal epithelial cells and from 0.96 μmol/L to 0.56 μmol/L in conjunctival epithelial cells(P=0.03). However, zeaxanthin did nothave significant effect on H2 O2-induced cell death in limbal or conjunctival epithelial cells.CONCLUSION: Zeaxanthin is an effective reagent in reducing the detrimental effect of UV-radiation and oxidative stress on ocular surface epithelial cells.
基金National Natural Science Foundation of China (No.81170816)Specialized Research Fund for the Doctoral Program of Higher Education (No.20113706110004)Qingjun Zhou is partially supported by the TaishanScholar Program of Jinan City, China (No.20081148)
文摘AIM: To determine the proliferative potential and the maintenance of stem cell activity in stored human limbal tissues, and correlate this with the preservation time, cell viability and the expression of stem cell markers. METHODS: Thirty limbal rims were split into 4 parts and stored in corneal preservation medium at 4 degrees C for 0, 1, 4, or 7 days. The limbal stem cell and mitotic markers P63, CK19, proliferating cell nuclear antigen (PCNA), and Ki67 were determined by immunohistochemical staining. The proliferative potential of limbal epithelial cells was assessed by cell viability, the ability of generating stratified epithelium, and colony forming assay. RESULTS: The stored tissues maintained limbal stratified structure to 7 days and exhibited comparable expression level of stem cell and mitotic markers. The proportion of viable cells decreased with the prolonged preservation time, while colony forming efficiency decreased from the 1st day and disappeared at the 4th day. When inoculated on amniotic membrane, the cells preserved for 1 day formed a stratified epithelium, while the cells from 4 days' preservation formed a discontinuous layer. CONCLUSION: The colony forming efficiency of limbal epithelial stem/progenitor cells decreased rapidly with the increasing preservation time, while the expression level of markers and capacity of forming epithelial monolayer on amniotic membrane decreased gradually. The limbal epithelial stem cells lost their function earlier than the lost expression level of stem cell markers. This may help us to better choose the appropriate preservation grafts for future limbal stem cell transplantation.
