动物造模,药效评价,角膜新生血管 z-bio 2024-07-18 459

　　Corneal neovascularization often leads to a decrease in corneal transparency, which is an important cause of blindness and a high-risk factor for rejection reactions after corneal transplantation. In clinical practice, CNV treatment remains one of the most challenging issues. The CNV animal model belongs to the category of induced disease animal models, which can artificially induce corneal neovascularization in animals through physical methods (such as thermal cauterization and corneal suture methods), chemical methods (such as alkaline chemical burns), biological methods (corneal pocket identification method), and surgical methods (such as heterologous corneal transplantation, intrastromal injection of bovine serum albumin, corneal stromal tumor tissue implantation, etc.), producing models similar to human diseases. The following mainly introduces the most commonly used method for corneal neovascularization research - corneal pocket identification method. The commonly used inducers include endotoxins, basic fibroblast growth factor, vascular endothelial growth factor, silica, etc. Taking the preparation of an animal model of corneal neovascularization induced by sustained-release endotoxins and bFGF sustained-release pills as an example, a brief introduction will be given.

　　The mechanism of endotoxin induced CNV is secondary to inflammatory response, and the inflammatory stimulus response is positively correlated with the induction of neovascularization. The process of endotoxin induced neovascularization may be regulated by bFGF factor secreted by macrophages, and amplified by other factors secreted by macrophages such as tumor necrosis factor - α and transforming growth factor - β.

　　BFGF is a single chain peptide containing 146 amino acids with complex functions. In a series of in vitro experiments, it has been proven to have effects on the differentiation, proliferation, migration, and chemotaxis of vascular endothelial cells. BFGF induced microvascular endothelial cells can produce a large amount of μ - PA, which plays a central role in the early stages of angiogenesis. μ - PA converts fibrinogen into enzymatically active fibrinolysin, which in turn activates collagenase, dissolves the basement membrane at the origin of neovascularization, and leads to

　　Endothelial cells enter the three-dimensional collagen matrix and form new capillary buds.

　　Preparation of sustained-release endotoxin polymer pills: Ethylene acetate vinyl ester polymer (Elvax) beads were washed with high-purity hexanal to spectrophotometric purity, and the polymer was dissolved in dichloromethane at room temperature to a final concentration of 10% (W/V). A certain proportion of E Mix Colin endotoxin with 10% Elvax and stir vigorously until a uniform suspension is obtained. The endotoxin Elvax suspension was dropped into an 8-well glass plate using a disinfectant dropper for polymerization. After complete polymerization, it was made into 1 cubic millimeter sized (1.3 ± 0.3) mg/pill. After UV disinfection, it was stored at -40 ℃ for future use.

　　Preparation of bFBF sustained-release pills: bFBF sustained-release pills are composed of a mixture of sustained-release agent poly (methyl methacrylate) -2-hydroxyethyl ester (Hydron) and a certain amount of inducer bFGF. A certain amount of sucralfate is added to the bFGF Hydron sustained-release pills to make a Hydron coated bFGF sucralfate sustained-release pill, which can effectively stabilize the biological activity of bFGF and slow down its release.

　　Corneal pouch production: All surgical steps were performed under sterile conditions. General anesthesia was administered via intravenous injection of 3% pentobarbital sodium into the rabbit ear vein (25mg/kg), and local eye anesthesia was administered with 1% lidocaine. A 1.5mm, 1/2 corneal thickness incision was made in the center of the cornea, and the 1/2 corneal pouch was separated at 6 points. The prepared sustained-release pill was implanted into a glass sleeve, and the pouch incision was reset for healing.

　　[Model features] CNV exhibits a dose-dependent relationship with local endotoxins. Significant corneal neovascularization growth can be achieved with a concentration of 15% endotoxin polymer, accompanied only by mild corneal edema, which can induce suitable CNV animal models. Excessive endotoxin concentration can cause significant keratitis, matrix turbidity, and easy fusion of neovascularization growth, affecting the accuracy of neovascularization measurement and calculation. Low concentration can result in insufficient induction force, which is not suitable for the requirements of the model.

　　The advantages of the sustained-release endotoxin polymer pill induced CNV model are: directional bundle growth of new blood vessels, stable repetition, significantly improved accuracy of measurement and quantitative analysis, and the ability to use a computer image analysis system for daily continuous dynamic quantitative measurement, which is convenient for comparison with the control group.

　　The advantage of the CNV animal model induced by bFGF sustained-release pills is that bFGF, as a direct stimulant of neovascularization, can exclude indirect angiogenic factors such as inflammation, which is of great significance for the efficacy evaluation of specific neovascularization inhibitors; Easy for quantitative analysis; Good stability and repeatability, and economical and practical.