Study Eye Cornea On The Microstructure,Endothelial Cell Activity And Gene Expression In Pigs

The purpose to study the biological characteristics of corneal cells in pig eyes and provide basic data for the possibility of corneal transplantation for using tissue and organ.The experiment was divided into four main parts :(1)Primary cell culture test of corneal endothelium in pig eyes.Comparing two culture methods of corneal endothelial cells in vitro,including for membrane adhesion method and double enzyme digestion method,the effective active function on corneal endothelial cells was obtained in porcine.(2)Study preservation and cryopreservation on corneal in pig eyes.Design preservation solution and cryopreservation solution were compared on porcine corneal endothelial cells with different temperatures(4℃,-20℃,-196℃)and different times(4h,6h,8h,12h).(3)Study the microstructure of mitochondria,ribosomes and endoplasmic reticulum on corneal stromal cells.Showing the basic data on nanometer animal biology(500 nm).The morphological characteristics were observed by electron microscopy from mitochondria,ribosomes and endoplasmic reticulum in corneal stromal cells.(4)m RNA expression of six major genes(Prdx-6,PITX2,VDAC3,ZP4,Na+/K+-ATPase,HTR1D)in corneal endothelial cells of pigs,can be used as a marker for the presence of corneal endothelial cells in pigs,and further distinguish corneal endothelial cells(CECs)from other foreign cells by the specific expression of genes.Results Showed:1.The endothelial cells were obtained by the method of uncovering the membrane and sticking to grow the wall slowly and adhere for a longer time.After 12 days,the cells were covered with the bottom of the petri dish,forming a single-layer pebble-shaped structure.After digestion with double enzyme,round endothelial cells with clear outline were obtained.The cells at 9~10d grew slowly and showed no obvious morphological changes,with a small number of spindle stromal cells.2.The corneal film was preserved with 4℃ preservation solution and stained.At different preservation periods: 4h,6h,8h and 12 h,the cells were clearly defined,closely connected,hexagonal or polygonal,and cell nuclei were not found.Corneal film preserved in-20℃ cryopreserved solution was stained,and the nuclei with different degrees of blue staining were found at 4h,6h,8h and 12 h after staining.4h,8h followed;There was a small amount of nuclear blue staining at 12 h.Corneal film stored in-196℃ cryopreserved solution,after staining,no blue-stained cells were found in 4h,6h,8h and 12 h.The cells were hexagonal or polygonal,but the CECs contour was not clear.3.The corneal stroma consists of laminated lamellar corneas,which the collagen fibers are perpendicular to the adjacent lamellar corneas.The spacing of collagen fibers remains periodic within each layer.Corneal stromal cells and their nuclei are located between adjacent lamella.There were a few mitochondria in corneal stromal cells,and there was no change in size and morphology.The damaged corneal stromal cells showed that vacuoles of different sizes,cell membrane rupture,organelles such as endoplasmic reticulum dispersed in the cells,and the nucleus was close to the membrane edge.4.The expression level of gene Prdx-6 was the highest in CECs from porcine corneal endothelial cells,while the expression levels of PITX2,VDAC3,ZP4 and Na+/K+-ATPase were lower in CECs,and almost no expression of HTR1 D.