Research Of Pressure-regulating And Mechanism Of Overpressure-induced Damage In Corneal Endothelial Cells

Objective:The purpose of the study was to analyze the morphology and structure features of rabbit corneal endothelial cells (RCEs)under different pressure gradient and variational regularity of their biological function through a pressure culture platform in vitro .The positive regulation action of normal intraocular tension pressure to the RCES activity is researched to provide theoretical evidence for 3D bionic culture system which was aimed to improve the culture quality of RECS ,and to construct engineering corneal endothelial cellular transplant membrane .The pathological bases and control links of corneal endothelial cells lesion under pathological high intraocular pressure is illuminated to provide experiment basis for endothelial cells protection in treatment of glaucomaMethods:Part1. to research the culture of endothelium cells under no-pressure Descemet membrane (DM ) was stripped from rabbit cornea under disseting microscope. DM with endothelium were incubated in 0.25%trypsase and 0.02%EDTA solution at and harvested purified RCE culture in vitro. Observated the cells morphous in inverted microscope, identified the cells by NSE when the cell fully confluent. The morphology of the endothelium was evaluated by HE. Flow cytometry to identify the sensitive of AnnexinV–PI.Part2. to research the bionic culture of endothelium cells under pressureWe designed and make a pressure -sustainded culture instrument with auto-filled pressure between 0-75mmHg,measured the lasting time of presetting pressure and fixed the time of auto-filled pressure .we put the culture board inoculated with heritable corneal endothelial cells into the pressure - maintenance culture instrument .With a preinstalled pressure of 2KP and cultured it continuously. Observated the cells morphous in inverted microscope, identified the cells by NSE when the cell fully confluent. The morphology and ultrastructure of the endothelium were evaluated by transmission electron microscopy and scanning electron microscope and HE. Used hematoxylin and eosin staining and alizarin red S-trypan blue staining to examine the activity of corneal endothelial cells, flow cytometry to identify the sensitive of AnnexinV–PI.Part4. to reseach the lesion mechanism of corneal endothelial cells under high pressureSimulating the clinical pathological process of glaucoma through biomimetic culture system in vitro,so the pressure is divided into :Acute pressure increase group(50mmHg),Chronic stress increased group(30mmHg),Pressure fluctuations Group(15mmHg~25 mmHg ~20 mmHg ~10 mmHg),Normal stress group(15mmHg)as the control group. Observated the cells morphous in inverted microscope. The morphology of the endothelium was evaluated HE.Used hematoxylin and eosin staining and alizarin red S-trypan blue staining to examine the activity of corneal endothelial cells, flow cytometry to identify the sensitive of AnnexinV–PI.RT- PCR analysis the expression of fas/ fasl in pressure and western -blotting detects the time-space curvilinear relation of p53 and Bcl-2; fluorescence observes the intensity and scope of cytc after the start of apoptosisResults:1.Large amount of purified corneal endothelial cells harvested after stripped from the DM,grow around the walls rapidly by link enzyme digestion and proliferated .They were merged into a single–linked cells after 3-4 days culture in vitro .The NSE antibody is expressed positive .HE staining and activity staining show good cell function.2.The growth activity of the corneal endothelial cells cultured under physiological pressure is good .the cells have strong stereo sense ,and are in flat shape with close inter cellular link. The cells are closer to the physical condition and there are seldom apoptosis cells cultured under the normal pressure. 3.The endothelial cells cultured under high pressure environment proliferated slowly. The cytoplast large and transparent cytoplasm contains plenty of granulation, and intercellular space was increased with the increasing of culture time. The expression of Anniex-v was strengthening and the proportion of apoptosis cells was significantly increased.4. The negative expression of fas/ fasl by RT-PCR, the expression of p53 significantly increased, and the expression of Bcl-2 significantly reduced under continuous high pressure. But the expression of p53 significantly reduced after preparation of the cells with inhibitor, and and the expression of Bcl-2 significantly increased. The expression of Cytc in cytoplasm marked increased of corneal endothelium cells in high pressure groups.Conclusion:1. Normal pressure bionic culturing environment could regulate the corneal endothelium cells function and change the physical shape which is closer to the body cells. It also supplys a new environment tempt platform for the construction of engineering rabbit corneal endothelial transplant membrane .2. The fluctuation of intraocular pressure is related to the corneal endothelium cell damage and resulted in apoptosis; high pressure could induce damage which was time- dose curve relation.3. The pathway of the corneal endothelium cells apoptosis was through the release of Cytc from the mitochondria and activated the intrinsic pathway.4. Caspase9 was as the drugs target of high pressure in glaucoma and provided the experiment bases on the protection for corneal endothelium cells.