Application of IL-6 to repairing of corneal epithelial injuries
A corneal epithelium, damage repair technology, applied in medical preparations containing active ingredients, epidermal cells/skin cells, artificially induced pluripotent cells, etc., can solve the biological function of corneal limbal stem cells and its activation mechanism is not very clear and other problems, to achieve the effect of strengthening the proliferation and clone formation of limbal stem cells, and promoting the damage and repair of corneal epithelium.
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Embodiment 1
[0022] Example 1: IL-6 promotes the clone formation ability of TKE2 cells
[0023] Apply TKE2 cell culture medium (Keratinocyte-SFM plus BPE (50 μg / mL) and rEGF (5 ng / mL): GIBCO, 10724-011) to routinely culture TKE2 cells, inoculate 1000 cells into 30 mm culture dishes, and pour Add IL-6 (20ng / ml), then culture under the conditions of 5% CO2 and 37 degrees Celsius, replace the new medium every 3 days, and add the above-mentioned concentration of IL-6 factor at the same time, fix the cells after 9 days and use crystal violet dyeing. Cell clone growth after IL-6 treatment see figure 1 . like figure 1 As shown, the addition of 20-50ng / ml IL-6 in the culture medium can effectively promote the formation ability of TKE2 cell clones.
Embodiment 2
[0024] Example 2: Effect of exogenous addition of IL-6 on the repair of corneal epithelial damage in normal mice
[0025] Twelve C57BL / 6 mice (6-8 weeks old) were randomly divided into control group and experimental group, and the epithelium in the central 3mm area of the right eye cornea of the mice was scraped using a 3mm trephine drill and an epithelial scraper. 5 μl PBS was injected subconjunctivally in the right eye, and 50 ng IL-6 (dissolved in 5 μl PBS) was injected subconjunctivally in the right eye of the mice in the experimental group. At 24, 48 and 72 hours after modeling, they were stained with sodium fluorescein and photographed under a slit lamp, and the mice in each group were observed. The repair of epithelial injury in mice, the representative slit lamp observation photos are shown in figure 2 . like figure 2 As shown, subconjunctival injection of 50ngIL-6 can effectively promote the repair of corneal epithelium damage.
Embodiment 3
[0026] Example 3: IL-6 inhibits the effect of miR-106a-5p expression level
[0027] TKE2 cells were routinely cultured with TKE2 cell culture medium (Keratinocyte-SFM plus BPE (50 μg / mL) and rEGF (5 ng / mL): GIBCO, 10724-011), and 10 5 Cells were inoculated into a 60mm culture dish, then cultivated for 24h at 5% CO2 and 37 degrees Celsius, added IL-6 (20ng / ml) to the dish, and incubated at 5% CO2 and 37 degrees Celsius for 0min , 15min and 30min later, the RNA of samples in each group was extracted and reverse-transcribed into cDNA, and the expression level of miR-106a-5p in each group was detected by real-time quantitative PCR. like image 3 As shown, IL-6 can significantly inhibit the expression level of miR-106a-5p when treated with IL-6 for 15 min and 30 min.
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