The Characteristics And Mechanism Of P16^ink4a Knockout Mice Neovascularization Model Induced By Corneal Sutures

Objective:By observing growing rule and similarities and differences which resulted in corneal neovascularization with suture technique about normal mice and the mice in loss of p16ink4a, and comparing the change of expression quantity and the relationship between corneal neovascularization and two groups in the process of corneal neovascularization. This paper will explore the role and mechanism of p16ink4a in the steady-state maintain of corneal neovascularization.Methods:(1) Build corneal neovascularization model of mice with suture technique. Select C57BL/6 mice as the control group, the mice in loss of p16ink4a as experimental group. The mice were general anesthetized, pressing central of corneal by corneal trephine lightly, forming corneal indentation diameter of 2mm for positioning stitches, making 3 stitches vertical indentation by 11-0 nylon line radially. (2) Observing the growth state of neovascularization by slit lamp. Discuss the differences about general time of happening, shape, density and scope by observing the growth states of corneal neovascularization with slit lamp from building model to suture until 14 days after operation. (3) To analysis the growth states of neovascularization and detect the expression of VEGF, its receptor and PEDF with immumofluorescence method. Materials were obtained by frozen section method after 5 days postoperatively to compare and analysis the distribution of neovascularization, density and VEGF, its receptor and PEDF, the variation of expression location and expression quantity by using VEGF-A, VEGF-R1, VEGF-R2, VEGF-R3 and CD31 to conduct double immunofluorescence staining between two groups. (4) To detect the expression of VEGF, its receptor and PEDF by molecular biology between two groups. Materials were obtained after 5 days postoperatively, to compare VEGF, its receptor and PEDF expression of mRNA, protein by using RT-PCR and western blot methods separately.Result:(1) Growth states of corneal neovascularization about control group and experimental group. The experimental group was more serious than normal group about blood vessel and corneal neovascularization with suture after operation, and neovascularization of experimental group was earlier than normal group in the aspect of invading of corneal stroma and getting to the suture position, in addition, neovascularization was than denser than control group at the same time. (2) The results of immunofluorescence. The density and position of corneal neovascularization:Cornea of the experimental group is significant edema by thicken. The expression of CD31’s positive arteries endothelial cell is more than control group, also the thickness of blood vessels increased after 5 days postoperatively. Cornea VEGF, receptor and expression of PEDF:The expression of VEGF-A, VEGF-R1, VEGF-R2 is high while PEDF is low, and VEGF-R3 is no significant difference after 5 days postoperatively. (3) Detect the expression of mRNA with RT-PCR:VEGF-A, VEGF-R1, VEGF-R2 increased significantly (p<0.01) while PEDF declined obviously, and the extent of variation is higher than control group (p<0.01) significantly after 5 days postoperatively. Also the expression of VEGF-R3 increased obviously after operation but there is no statistical significance between two groups. (4) Detect expression of protein with Western blot:After 5 days postoperatively, compared the experimental group and control group, the degree of expression about VEGF-A, VEGF-R1 increased significantly while PEDF declined. In addition, VEGF-A/PEDF of the experimental group of more significant than control group.Conclusion:The mice whit p16ink4a knockout is more significant than control group in speed, length and density of corneal neovascularization after induced by corneal stitch resulting. At proliferative phase of blood vessels, VEGF-A of mice whit p16ink4a knockout is higher while PEDF is lower, also VEGF-A/PEDF is more obvious than control group. pl6ink4a may maintain the steady state of blood vessels of corneal limbal by moderating the relative expression levels of VEGF and PEDF.