| PurposeCulture human conjunctival goblet cells in vitro,identify the morphological structure and the properties;Culture normal human limbal and conjunctival epithelial tissue on the amniotic membrane,observe the differences between them;Discussion the influence ofγ-secretase inhibitors on cultured human conjunctival goblet cell proliferation and specific protein expression of CK7;Observation of amniotic membrane as a carrier of recombinant human conjunctival epithelium and the difference between normal conjunctival epithelium.MethodsPart1 Culture human conjunctival epithelial cells in vitro and identification1. Culture human conjunctival epithelial cells in vitroTake the human conjunctiva from eye bank,cultured human conjunctival epithelial cells isolated for culture within 6 hours by tissue culture method.2. IdentificationBy AB-PAS staining to identify goblet cells initially,and estimate its density; by immunocytochemistry staining of suspended cells Muc5Ac expression, identification of goblet cells; by immunofluorescence staining CK7 expression detection and identification of goblet cells.Part2 The difference of normal limbus and conjunctival epithelium cultured on amniotic membrane.To take limbus and conjunctiva from eye bank,culture corneal tissue on amniotic membrane by digestion,and the conjunctival epithelium cultured with tissue culture on amniotic membrane carrier; compare the two differences by PAS staining. Part3 Cultured human conjunctival goblet cells byγ-secretase inhibitors induce differentiation and related testing.1. Induction of differentiation of conjunctival goblet cellsPrimary cultured conjunctival epithelial cell density reached 30 to 40%, the randomly selected four holes were added with 100nM, 200nM, 400nMγ-secretase inhibitors of the culture medium, a hole as a negative control, observation of 72h. Conjunctival goblet cells in vitro byγ-secretase inhibitors detected after induction of differentiation.2. Primary cultured human conjunctival epithelial cells after 72h of drug stimulation, the use of AB-PAS staining to compare the same time, after stimulation with different concentrations of drugs difference between goblet cell density; extracted from the same time, different concentrations of drugs to stimulate future generations conjunctival epithelial cells of total RNA, cDNA underwent reversed into a real-time fluorescence quantitative PCR (RT-Q-PCR) test, the CK7 mRNA expression observed at the same time, different concentrations of drugs to stimulate the group changes in the conjunctival epithelial cells; extractnegative Control and 200nMγ-secretase inhibitors after 72h stimulation of human conjunctiva epithelial cells, total protein, Western-Blot test line CK7 protein expression in human conjunctival epithelial cells by immuno- -histochemistry.3. Statistical analysis: The results of the analysis of variance (Analysisi of Variance, ANOVA) test was used for statistical analysis and comparison of different concentrations ofγ-secretase inhibitors stimulate the group and negative control group differences, with P <0.05 as a difference statistically significant.Part4 Amniotic membrane as a carrier recombinant conjunctival epithelial compared with normal conjunctival epithelium.To take the normal conjunctiva from eye bank and culture on amniotic membrane carrier and use PAS staining, conjunctival epithelium of normal paraffin sections with PAS staining and compared the difference between the two. ResultsPart1 Culture human conjunctival epithelial cells in vitro and identification1. Tissue culture cells cultured normal human conjunctival epithelium, the next day, cells can be seen within 6 hours to move out from the surrounding tissue, cells adherent growth, 15 days can be covered dish, we can see more goblet cells in suspension.2. Identificationa AB-PAS staining showed double staining: the cytoplasm was blue and red particles are the goblet cells can be identified in vitro cultured human conjunctival epithelial cells with goblet cells.b Immunocytochemistry showed that: The primary cultured human conjunctival epithelial cells suspension cells of mouse monoclonal antibody Muc5Ac stained positive for the goblet cells. Proof of cultured human conjunctival epithelial cells in the cell suspension containing goblet cells.c Immunofluorescence results showed that: The primary cultured human conjunctival epithelial cells of mouse monoclonal antibodies CK7 staining is positive for the goblet cells. Can prove that cultured human conjunctival epithelial cells contain goblet cells.Part2 The difference of normal limbus and conjunctival epithelium cultured on amniotic membrane.PAS staining showed that: cultured on amniotic membrane with limbal epithelial cells and conjunctival epithelial cells were goblet cells with positive staining; limbal epithelial cells in the goblet cells in conjunctival epithelial cells than low density, and low differentiation.Part3 Cultured human conjunctival goblet cells byγ-secretase inhibitors induce differentiation and related testing.a. Inverted microscope: Add toγ-secretase inhibitor group goblet cell density higher than the negative control group, which 200nM group increased the most obviously.b. AB-PAS staining showed double staining:γ-secretase inhibitors added 72h after the stimulation of epithelial cells in human conjunctiva goblet cell density higher than the negative control group; drugs to stimulate the group 200nM group, the highest density of goblet cells.c RT-Q-PCR results showed that: 100nMγ-secretase inhibitors stimulate the group and negative control group differences in the expression of CK7mRNA significant.d Western-blot results showed that: The results showed that the expression of CK7 protein 200nM group than in the negative control group was significantly increased. Part4 Amniotic membrane as a carrier recombinant conjunctival epithelial compared with normal conjunctival epitheliumPAS staining showed that: the recombinant amniotic membrane as a carrier in the goblet cells in conjunctival epithelial cells than normal density of conjunctival epithelial goblet cell density in the low; the latter embedded in PAS staining goblet cells in conjunctival epithelium, the former PAS staining goblet cells located in the membrane surface, easy to fall off.Conclusions1. Human conjunctival goblet cells can be cultured in vitro and in vivo properties with; cultured goblet cells in suspension growth.2.γ-secretase inhibitors in vitro can be induced conjunctival epithelial precursor cells to differentiate into goblet cells and goblet cells can promote gene expression of specific proteins CK7.3.γ-secretase inhibitors can induce cells to differentiate into goblet conjunctival epithelial cells and can promote mucin and goblet cell-specific marker gene expression.4. To use amniotic membrane as the carrier,there are some problem such as goblet cell density is low, the location is different from the normal conjunctival epithelium,so need to find a new carrier or method of inducing differentiation of recombinant conjunctival epithelium. PurposeCorneal ring placed in medium-term solution in preservation of cornea at different times, through the detection cell volume, cell activity, stem cell associated markers, cell proliferation and differentiation analysis of medium-term preservation solutions on corneal limbal stem cells.Methods1. Pretreatment of corneal ringTake a total of six corner rings from eye bank, under the microscope peel off the corneal endothelium and the iris,then each divided into 3 parts, medium-term preservation solution were placed in the cornea 1d, 4d, 7d.Digestion limbal epithelial into single cell suspension.2 Different limbal epithelial cells detected by the total cell count, mean cell volume calculated;The cells were seeded on the 3T3 feeder layer, culture and Giemsa staining observed, based on cell colony formation ability. The limbal stem cells in them were analyzed by flow cytometry.By immunofluorescence technique to detect the relevant stem cell marker expression. The cells were seeded on amniotic membrane, by HE staining cells in histopathology complex layers forming ability.Results1. Detection of cell volume With the limbal stem cells stored in the medium-term preservation solution time, the cell volume decrease.2. Cell activity With the limbal stem cells stored in the medium-term preservation solution time, the cell activity was reduced.3. CFEWith limbal stem cell preservation solution in the medium term time saved, reduced cell CFE.4. Detection of limbal stem cell associated marker P63With the limbal stem cells stored in the medium-term preservation solution time,P63 expression showed an increasing trend.5. Antigen detection (immunofluorescence method)With the limbal stem cells stored in the medium-term preservation solution time, limbal stem cell associated markers P63, ABCG2 expression and so significantly reduced.6. Histopathological (HE staining)With the limbal stem cells stored in the medium-term preservation solution time, the lower the ability of cells to form multiple layers.Conclusions1.Application of mid-term solution in preservation of corneal limbal tissue in which the short term to maintain the activity of limbal stem cells and function, but with the extension of storage time, the activity of limbal stem cells and functional decline in different degrees.2.The function of limbal stem cell and the expression of its makers have no direct relation: the expression of P63 does not mean the ability of colony forming or form monolayer in the amniotic membrane. |
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