| Purpose1. Have a discussion on the feasibility of separate culturing of rabbit mesenchymal stem cells in vitro; build mesenchymal corneal stroma; and observe an ultra-structure of corneal stroma;2. Have a discussion on the influence of clear supernatant liquid of mesenchymal stem cells on growth of keratocytes cultured in vitro and analyze the reason for it.3. Have a discussion on the curing effect of mesenchymal stem cells on chemical burns of cornea for rabbits, and make a detection of expression levels for related cytokine in curing process; discuss mechanisms under which corneal stroma plays a curing role.Methods1. Separate bone marrow cells of young rabbit with adherence method and culture them in vitro, and continue to subculture and purify the cells gained; make a morphological observation of the cells under an inverted microscope, take photos and make a comparison of culture effect of inoculated cells under different concentrations; perform experiment on cell differentiation to adipocytes and osteoblasts and conduct evaluation, observe differentiation ability of mesenchymal stem cells; make a detection and identification on the cells surface with CD29, CD44, and CD45, and observing the purity of the cells gained;detect the clone ability of mesenchymal stem cells; induce multilayer growth of rabbit mesenchymal stem cells cultured in vitro with Vitamin C, and build corneal stroma of mesenchymal stem cells, then make a histological identification of the HE chromosome.Observe tissue components and cell states of cell patch for BMSCs under scanning electron microscope and transmission electron microscope; detect cytokine expression of cell culture fluid supernatant in vitro under different concentrations, such as IL-2, MMP-9,VEGF, IL-10, TSP-1 and TSG-6 with ELISA method.2. Isolate and culture the corneal stromal cells of rabbit to its logarithmic growth phase,and use serum-free DMEM(Serum-free FCM), culture medium(FCM) with 10% FBS DMEM and BMSC supernatant(MSC medium) to culture corneal stromal cells. MTT,scratch test and cell cycle detection should be used to test the effects of these 3 culture media. ELISA should be used to test the cytokine expression of IL-2, IL-10, TSP-1 and TSG-6 under 3 conditions.3. After the establishment of the model of chemical burns of cornea, 72 New Zealand white rabbits were divided into experimental group and control group randomly. On the7 th, 14 th and 21 st day after the operation respectively, the integrity of the corneal epithelium, matrix transparency and angiogenesis were examined by using slit lamp microscope, and after the observation and photograph, 3 experimental animals were chosen as a group randomly and tested for factors such as VEGF, MMP-9, IL-2 and S100A8 through Westernblot test, factors such as EGF, MMP-9, S100A8, IL-2, TSP-1, TSG-6 and IL-10 were tested through RT-PCR and ELISA tests. HE-staining of tissue section and immunofluorescence were used to detect the cytokine expression.ResultsPart I1.1 Cell patch of corneal mesenchymal stem cells was successfully constructed; 24 h full medium primarily cultured through BMSCs was effective. Relatively depurated BMSCs were produced from the third generation of cells with good refractivity of their edges which can be used in subsequent experiments; for adipogenesis experiment, BMSCs were stained by oil red, no lipid droplet formed in the cells, and the stain result was negative; for osteogenesis induced experiment, osteoblast revulsant was used in the cultivation process.On the 21 st day, alizarin red staining was observed under the microscope: the cells grew with many layers with overlapping orders. Individual cells with spindle morphology disappeared and most cells were in polygonal shape. Calcareous sediments were found in intercellular substance, alizarin red staining showed calcified node, and the stain result was positive; Flow cytometry instrument testing BMSCs CD29 positive rate was 93.7%, CD44 was 93.4%, CD 45 positive rate was 0.32%. 2 x105 cells/hole vaccination, failure to form membrane surface sediments, can be clearly observed in cell morphology, cell edge refraction, forming the corneal stroma activity;1.2 synaptic transmission electron microscopy(TEM) observation visible cells and secretory granule and abundant cytoplasm with a large amount of collagen bundles, cells in good condition. IL-2, MMP-9 and VEGF in 2 x 105 density expression quantity is lower than other cells inoculated density; IL-10, 1 TSP – 1 and TSG-6 in 2 x 105 density expression quantity is higher than other cells inoculated density.Part IIFCM to promote cell migration, cell migration, MSC medium and serum- free FCM compared with FCM inhibition corneal stromal cell migration, MSC medium significantly influence stromal cells proliferation compared with FCM. ELISA test results in the MSC medium cytokines IL-10, the TSP-1 and the number of TSG-6 expressions is significantly higher than FCM and the expression of cytokines in Serum- free FCM Serum.Part III3.1 slit lamp observation: group 7 days after the visible corneal smooth, epithelium has been completed; 14 days after the visible corneal transparency and no scar tissue formation;21 days after the experimental BMSC observations, has fully transparent cornea, ocular surface is smooth, epithelial integrity and treatment effect is obvious.3.2 histopathologic observations: the postoperative day 7 visible cell membrane is loose,corneal epithelium has been completed. Day 14 cell membrane has been largely and corneal stroma integration; 21 days after the operation, the cell membrane has completely with corneal integration.3.3 Westernblot experiment results: VEGF, MMP-9, IL-2 and S100A8 factor in 7 days, 14 days and 21 days normal group and BMSC treatment group expression level is low, but the control group in the expression quantity obviously increased;3.4 detect the cytokine expression results: in the normal rabbit corneal stromal each gene expression level is low; in the control group(plate layer removing corneal trauma) VEGF,MMP-9, S100A8 and IL-2 were significantly increased; BMSC group compared with control group, the TSP-1, TSG-6 and IL-10 expression varied significantly increased.3.5 detect the cytokine expression : immunofluorescence techniques: the control group in the cornea invasion of CD4 gradually increased significantly higher than the MSC Treatment group and normal group; Control group in the cornea IL-2 and MMP-9expression quantity are gradually increased, significantly higher than the MSC treatment group and normal group;3.6 ELISA to detect cytokine results: EGF, MMP-9, S100A8 and IL-2 in the control group,high expression and 3 weeks to express the highest amount. The TSP-1, TSG-6 and IL-10 in experimental group expression of BMSCs diaphragm processing quantity is higher, the effectively inhibit vascularization, inflammation and scar tissue.ConclusionConstructing in-vitro BMSCs cell sheets has observable therapeutic effects on chemical burns of cornea. It can promote epithelialization of cornea and maintain the transparency of corneal stroma. It can also inhibit vascularization, inflammation and scar tissues effectively.Its mechanism of action might be related to the expressions of up-regulation TSP-1, TSG-6and IL-10, down-regulation VEGF, MMP-9, S100A8 and IL-2. |
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