Effects And Mechanisms Of Proinflammatory Factors On The Activity Of Corneal Epithelial Stem Cells

Purpose:To study the influence chronic inflammation factors on activity and aging of corneal epithelial stem cells, and further explore its action mechanism.Methods:1.The establishment of corneal epithelial stem cell model in vtro:a murine limbal/corneal epithelium-derived progenitor cell line (TKE2,), was incubated in keratinocyte serum-free medium with BPE and EGF; change to keratinocyte serum-free medium with only EGF without BPE for 1 days berore experiment; the cells were treated with 10 ng/ml of IL-1β or TNF-a for 8 days; experiments as follow:(1) The influence of inflammatory factor on the proliferation and differentiation of corneal epithelial stem cells:staining immunofluorescence detects the change of fluorescence intensity of Ki67、importin13、ck3/12、involucrin in each group treated by different inflammatory factors.Collecting samples,detecting the expression of each groups of importm13、Bmil by Western blot.(2)The influence of inflammatory factor on the aging of corneal epithelial stem cells:staining immunofluorescence and Western blot detect the change of fluorescence intensity of p16 in each group treated by different inflammatory factors, detecting ROS level in cells by DCFH-DA.Collecting samples,detecting the expression of each groups of p16 by Western blot.(3) The influence of inflammatory factor on the STAT3 signal pathway:staining immunofluorescence and Western blot dectect the change of fluorescence intensity of STAT3 in each group treated by different inflammatory factors.2.The establishment of corneal epithelial scrape model, experiments as follow:(1) Study on the repair of corneal epithelial injury in mice:Sodium fluorescein staining after 24h、48h、72h for corneal epithelial curettage. Take photos by Slit lamp camera,and calculat the Corneal epithelial defect area by Image J.(2) Effect of p16ink4a deficiency on proliferation of corneal epithelial cell and stem nature of stem cells:after murine corneal epithelial curettage for 48h, take the eyes, making the 8 μm frozen section, staining immunofluorescence,,to detect the change of Ki67、ABCG2. Collecting mice corneal epithelium from normal C57BL/6 mice and p16ink-4a deficient mice, detecting the expression of Bmil by Western blot and the expression of p63、ABCG2 by real time PCR.(3) Effect of p16ink4a deficiency on STAT3 signal pathway:after murine corneal epithelial curettage for 48h, staining immunofluorescence and Western blot detect the change of expression of the normal C57BL/6 mice and p16ink-4a deficient mice STAT3.(4) Effect of p16ink4a deficiency on the expression of corneal epithelial cell anti-oxidation factor:after murine corneal epithelial curettage for 48h, Western blot detects the change of expression of the normal C57BL/6 mice and p16ink-4a deficient mice SOD2. PCR detects the change of expression of the oxidation resistance genes.Results:1.The treatment of IL-P and TNF-a can inhibit proliferation and stem nature of TKE2 cell,promote its differentiation. After inflammatory molecules process TKE2 cells, compared with normal control group, the expression of Ki67 was significantly decreased, and the expression of stem cell markers importin13, Bmil was significantly reduced, and the difference was statistically significant (P<0.05). While the expression of the corneal epithelial differentiation marker ck3/12, involucrin was increased significantly.2.The treatment of IL-β and TNF-α can inhibit aging of TKE2 cell. After inflammatory molecules process TKE2 cells, compared with normal control group, the expression of p16 was significantly decreased, and the expression of stem cell markers importin13, Bmil was significantly reduced, and the difference was statistically significant (P<0.05). While accumulation of ROS.3.The treatment of IL-p and TNF-a can inhibition the activation of STAT3. After inflammatory molecules process TKE2 cells, compared with normal control group, the expression of p-STAT3 was significantly decreased, the difference was statistically significant (P<0.05).4.The lack of p16ink4a can promote repairing the corneal epithelial injury. after corneal epithelial curettage, epithelial regeneration ability in p16ink4a deficient mice is stronger, and promote the healing rate of mouse corneal epithelial compared with normal C57BL/6 mice significantly.5. The lack of p16ink4a can promote the proliferation of corneal epithelial cells and the activity of stem cells. Corneal epithelial of p16ink4a deletion in the process of wound repair, the expression of Ki67 was significantly decreased, and the expression of ABCG2,Bmil and p63 was significantly decreased.6.The lack of p16mk4a activated STAT3 signaling pathway of corneal epithelial cells. After murine corneal epithelial curettage for 48h, the fluorescence intensity of p-STAT3 in the corneal epithelium of p16ink4a mice Stronger than that in the corneal epithelium of C57BL/6 mice, the expression of p-STAT3 in the corneal epithelium of p16ink4a mice more than that in the corneal epithelium of C57BL/6 mice, the difference was statistically significant (P< 0.05)。7.The lack of p16ink4a activated the expression of corneal epithelial cell anti-oxidation factor. After murine corneal epithelial curettage for 48h, the expression of SOD2 in the corneal epithelium cells of p16mk a was significantly decreased, and the expression of SOD2, Catalase, Hmox1, GCLC and TXN was significantly raised.Conclusions:The presence of inflammatory factor as IL-1β、TNFα causes that the corneal limbus stem cells lose activity and appear aging characteristics, the expression of p16 is significantly decreased, the expression of STAT3 is significantly inhibited. While the lack of p16ink4a can promote repairing the corneal epithelial injury, promote activation of STAT3, and strengthen the antioxidant capacity of corneal epithelium. Therefore chronic inflammation may regulate signaling pathways of p16ink4a/STAT3 to affect the activity of corneal epithelial stem cells.