| Objective: To study the methods of detaching, culturing and multiplying rabbit bone marrow mesenchymal stem cells in vitro. To observe the survival, migration and differentiation of the rabbit mesenchymal stem cells after transplanted onto the alkaline burn rabbit cornea and to investigate the possibility of differentiating into corneal epithelial cells in vitro. That will give a new choice for treating the functional disorder or deficiency of corneal epithelial cells and limbal stem cells.Methods: After anesthetization, left eye of each 18 New Zealand rabbits (2.0～2.5 kg) was alkali burned by circum-type filter-paper soaked in 1mol/L NaOH for 1 min under sterile conditions. Human amniotic membrane was obtained at the time of cesarean section. Amniotic epithelium and endothelium were removed by Trypsin- EDTA treatment. The rabbit bone marrows were aspirated from the iliac crests. The alls were added slowly to equi-capacity Percoll solution (density 1.094g/ml) and centrifugated 25 minutes at 2500 rpm. Subsequently, the grey layer solution was aspirated out which contains mononuclear cells. Cells were seeded in LG-DMEM supplemented with 10% fetal bovine serum ( FBS ). Then the cell was observed and the cell growth curve was draw. The cell surface antigens of the passage 3 cells which contain CD29, CD45, CD34 were detected with flow cytometry. The passage 3 cells were cultivated on the human amniotic stroma carrier. 5-Bromo-2-deoxyuridine (BrdU density 10μmol/L) was added into nutritive medium before the transplantation. These 18 New Zealand rabbits were randomized into three groups: control group,simple amniotic stroma transplantation group and rabbit mesenchymal stem cells transplantation group. Transplantation was taken 1 month after alkali burn. The corneal changes were observed carefully. The corneas were obtained from eye balls after 4 weeks, and the corneal morphology were analyzed by clinical observations and hematoxylin-eosin staining. The survival and differentiation of rabbit mesenchymal stem cells were observed by BrdU and cell keratin AE5 monoclone antibody immunohistochemical examination.Results: The cells which isolated by Percoll density centrifuge from rabbit bone marrow were adherent to the flasks and grew as spherical shape 24 hours. After 3 days, cells which proliferated rapidly integrated colony and grew as typically fusiform shape. Primary cells reached 80%～90% confluence in about 12 days. The result of flow cytometry showed that the passage 3 cells typically expressed CD29, while CD34 and CD45 of them were negative. The rabbit eyes which burned by circum-type filter-paper soaked in NaOH showed corneal opacity, corneal neovascularization and scarring. Impression cytology examination indicated corneal surface conjunctivalization. The corneas which received the cultured rabbit mesenchymal stem cells transplantation were transparent. Histopathological examination revealed the structural character of normal corneal. The epithelium lined up in order and were adherent to the stroma. Anti-BrdU and anti-AE5 positive epithelial cells were found and no signs of goblet cells and neovascularization. Suggest that these cells can express normal cornea-specific keratin. On the contrary, for the rabbits which only received simple amniotic stroma transplantation, the corneas were opacity moderately. The Histopathological examination revealed the disorganized epithelium with goblet cells among them, under which there were inflammatory cells and neovascularization in the stroma, no anti-BrdU and anti-AE5 positive epithelial cells were found. However, the corneas of the control group were opacity obviously, distinct neovascularization were observed in the center of cornea. In the HE stained corneas, there were a lot of goblet cells, inflammatory cells and neovascularization, and no anti-BrdU and anti-AE5 positive epithelial cells were found in immunohistochemical stain.Conclusion: By using Percoll solution (density 1.094g/ml) density gradient centrifugation and adherence screening, the rabbit mesenchymal stem cells could be successfully isolated in vitro. Through burning cornea by circum-type filter-paper soaked in NaOH, could establish limbal stem cell deficiency model. The rabbit mesenchymal stem cells could survive and differentiate into corneal epithelium after transplanted onto the corneal stroma. The rabbit mesenchymal stem cells transplantation could decrease corneal conjunctivization after alkaline burn better than simple amniotic stroma transplantation. We believe that transplantation of mesenchymal stem cells represents an effective technique for ocular surface reconstruction in patients with severe limbal stem cell deficiency. |
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