| ObjectiveTo analyze the morphology and structure features of rabbit corneal endothelial cells (RCEs)cultured under different pressure gradient and variational regularity of their biological function through a pressure culture platform in vitro .To investigate the positive regulation action of normal intraocular tension pressure on the RCEs activity, and provide theoretical evidences for 3D bionic culture system which aimed to improve the culture quality of RECs and construct engineering corneal endothelial cellular transplant membrane. The pathological bases and regulation of corneal endothelial cells lesion under pathological high intraocular pressure is illuminated to provide experimental basis for endothelial cells protection in treatment of glaucoma.MethodsPart1.to explore an improved method of culture corneal endothelial cellsDescemet membrane (DM) with endothelium was stripped from rabbit cornea. DM was incubated in trypsin and EDTA solution, purified RCEs were harvested and cultured in vitro. Morphous of cells were observed under inverted microscope, cells growth curves were described and the cells were identified by NSE when cells reached full confluence. The activity of corneal endothelial cells was measured by hematoxylin and eosin staining and alizarin red S-trypan blue staining, the sensitive of AnnexinV–PE was identified by flow cytometry. The morphology and ultrastructure of the endothelium were evaluated by transmission electron microscopy and scanning electron microscope.Part2.to investigate the bionic culture of endothelium cells under pressureA pressure-sustained culture instrument with auto-filled pressure between 0-60mmHg was designed, the interval of auto-filled pressure was adjusted with the duration of presetting pressure. Corneal endothelial cells were cultured under a pressure of 2KP(14.66mmhg). The morphology of cells were observed, the time of cells reaching full confluence and cells double–growth curves were recorded. After the cells reached confluence, the cells activity were evaluated by alizarin red-S-trypan blue staining. The site and intensity of cell link protein ZO-1 were determined by laser confocal microscope and the expression of caspase-3 was evaluated by immunofluorescence technique and the ratio of AnnexinⅤ-PE positive cells was identited by flow cytometry technique.Part3 to construct an engineering corneal endothelial cells transplant membrane by bionic culture under 3D pressureEpithelial cells on amniotic membrane were removed by digested with trypsin, purified rabbit corned endothelial cells cultured on the basement membrane which was kept as carrier. To construct a engineering corneal endothelial cells transplant membrane, we put it in a pressure culture system for a 3D bionic culture. The tissue structure and morphology of constructed transplant membrane were observed under electronmicroscope and HE staining, the expression of ZO-1 was analyzed by immunofluorescence. .Part4 to reseach the mechanism of the impairment of corneal endothelial cells under high pressureThe pressure was set at 3.2kp～4.6kp (24.06mmhg～34.59mmhg),5.4kp～6.8kp(40.60mmhg～51.29mmhg). After cultured under high pressure, morphology of corneal endothelial cells and change of their proliferation activity were observed, and evaluated the relevance between biological activity of corneal endothelial cells and pressure magnitude, lasting time, to determine the early expression of caspase-3 and AnnexinⅤ.Results1.Large amount of purified corneal endothelial cells were harvested by stripped and digested DM, grew and proliferated rapidly after adherence. Cells merged into monolayer after 3-4 days culture in vitro. The expression of NSE was positive. HE staining and activity staining showed good cells'function. 2.The growth activity of the corneal endothelial cells cultured under physiological pressure was good, and showed strong stereo sense and flat shape with close inter cellular link, ZO-1 protein on cell membrane and junctional zone was highly expressed, closed to the endothelial cells growing under physiologic condition of organism. There was no obvious apoptosis under physiological pressure .3.Under pressure culture system, corneal endothelial cells grow rapidly and proliferate after inoculated to the cell-shed amniotic membrane and merged into a monolayer after 3-4 days. Their morphology and growth density were superior to those cultured in pure culture plate. HE Staining and election microscope showed that the engineering endothelial cell transplant sheet had similar tissue structure with physiologic corneal endothelial tissue.4.The endothelial cells cultured under high pressure grew slowly with large cytoplast and transparent cytoplasm which contained plenty of granulation, increased intercellular space. With the increasing of culture time, the expression of caspase-3 and AnnexinⅤenhanced and the ratio of apoptosis cells increased remarkably.Conclusion1.The method of DM stripped from rabbit corneal combining trypsin digestion can enhance the efficiency of harvesting and culturing corneal endothelial cells. Purified and large amount of rabbit corneal endothelial cells can be harvested by this method. Cells grow well with fewer epithelial cells and ground substance. It provides stable seed-cell source for the construction of engineering rabbit corned endothelial transplant membrane.2.Low pressure bionic culture plays a positive role on the regulation of the function of rabbit corneal endothelial cells. It can improve the conjunction of rabbit corneal endothelial cells in vitro. It also supplies a new environment platform for the construction of engineering rabbit corneal endothelial transplant membrane.3.Under low-pressure 3D bionic culture system, engineering endothelial cell transplant membranes which similar to physiologic endothelium in cell density, morphology and tissue structure can be constructed to make a prophase technique for corneal endothelial cell transplant studies.4.Corneal endothelial cells were damaged by high pressure, apoptosis may be involved in .It shows a time-dose–effect curve. Caspase-3 and AnnexinⅤare key factors in apoptosis. |
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