OBJECTIVE: To study the relationship between the postmortem interval (PMI) and the changes of DNA content in rabbit's cornea epithelial and endothelium. METHODS: Using the computerized image analysis to measure the DNA content in the cornea epithelial and endothelium of 105 rabbit's at every six hours during 72 hours after death. RESULTS: The degradation rate of DNA in nuclear has an apparent relationship in 72 hours after death of the rabbits. CONCLUSION The determination of the quantity of DNA in cornea nuclear is a new method to estimate the PMI accurately.
Animals ; DNA/analysis* ; Endothelium, Corneal/chemistry* ; Epithelium, Corneal/chemistry* ; Female ; Forensic Medicine ; Male ; Postmortem Changes ; Rabbits ; Signal Processing, Computer-Assisted ; Time Factors

Un-transfected rabbit corneal endothelial cells (RCECs) were cultivated, using chitosan blend membrane 4ha (chitosan-hyaluronic acid), 631ha (chitosan-hyaluronic acid) and 631s (chitosan-chondroitine sulfate) as scaffold carriers. Their biocompatibilities were studied in regard to cell adherence, morphological changes, growth status and monolayer forming abilities. The results indicated that RCECs cultivated on 4ha and 631ha carriers tended to be aggregated and even desquamated to some extent in local areas, and even more severely on 631ha carrier. And the RCECs cultivated on 631ha carrier could form almost a monolayer 48h later, and those on 4ha carrier could not. Contrarily, the RCECs cultivated on 631s carrier were evenly distributed and were in good status of growth with a good adherence and fibroblast-like morphology which could form almost a monolayer 48h later. And a complete monolayer was formed and was tightly attached to the 631s carrier 72h later. From the above results, it can be concluded that 631s carrier is most probably an ideal scaffold carrier for RCEC cultivation. 631s carrier may have the potential for use in the development of tissue-engineered rabbit corneal endothelium.
Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; Cell Culture Techniques ; Cell Line ; Cells, Cultured ; Chitosan ; chemistry ; Endothelium, Corneal ; cytology ; Hyaluronic Acid ; chemistry ; Membranes, Artificial ; Rabbits ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

OBJECTIVETo investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application.

METHODSTotal RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF cDNA was obtained with reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E. coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were observed under an inverted phase contrast microscope, a scanning electron microscope and a transmission electon microscope, respectively.

RESULTSPDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a (+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells.

CONCLUSIONSThe construction of recombinant prokaryotic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the proliferation of cultured cat corneal endothelial cells.

Animals ; Cats ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Endothelium, Corneal ; cytology ; drug effects ; Humans ; Immunohistochemistry ; Phosphopyruvate Hydratase ; analysis ; Protein Folding ; Proto-Oncogene Proteins c-sis ; chemistry ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Wound Healing ; drug effects

BACKGROUNDTrabecular meshwork (TM) cell volume may be an important determinant of aqueous humor outflow in the eye. This study aimed to evaluate the role of HepII domain peptides V on corneal permeability, corneal endothelial cells, intraocular pressure (IOP) and morphology of trabecular meshwork in rats.

METHODSThe IOP of rat eyes was measured before and 3, 5, 7 and 8 hours after topical delivery of HepII domain peptides V through intracameral injections. The peptide's concentration in aqueous humor was assessed by high performance liquid chromatography (HPLC). The shape and density of endothelial cells were observed by laser confocal microscopy 8 hours, 3 and 14 days after intracameral injections of HepII domain peptides V. The morphological changes in TM of rat eyes were assessed by transmission electron microscopy (TEM).

RESULTSIntracameral injection of HepII domain peptides V significantly (P < 0.001) decreased IOP by (5.71 ± 2.10) mmHg in rats at 5 hours after injection. There were no obvious changes of the shape and the density of corneal endothelial cells. In addition, morphological changes in the TM of rats were observed including the expansion of intercellular spaces in the juxtacanalicular meshwork, removal of extracellular material, cellular relaxation, and cytoskeleton reorganization.

CONCLUSIONSHepII domain peptides V could not penetrate cornea and was safe to corneal endothelial cells. HepII domain peptides V could significantly decrease IOP in rat probably by disorganizing actin cytoskeleton and cell-junction in the TM.

Animals ; Chromatography, High Pressure Liquid ; Cornea ; cytology ; drug effects ; ultrastructure ; Endothelium, Corneal ; drug effects ; ultrastructure ; Female ; Fibronectins ; chemistry ; pharmacology ; Intraocular Pressure ; drug effects ; Male ; Microscopy, Confocal ; Microscopy, Electron, Transmission ; Rats ; Rats, Sprague-Dawley ; Trabecular Meshwork ; drug effects ; ultrastructure