动物造模,药效评价,葡萄膜炎 z-bio 2024-08-09 409

　　1. Model making method: Wistar rats weighing around 200g.

　　(1) Preparation of endotoxins: Vibrio cholerae 18001 strain, classical biotype, Ogawa serotype. ① The purified Vibrio cholerae species that have been identified will be first transferred from the inclined surface of the Houttuynia cordata to the Houttuynia broth medium and cultured overnight at 37 ℃. On the second day, expand the Houshi flat culture to 20 bottles and incubate at 37 ℃ for more than 4 hours. After the pH stabilizes, add formaldehyde to inactivate and centrifuge to harvest the bacteria. Store at 20 ℃. ② The separation and purification of LPS were carried out following the steps of West2phal and Jann's phenol water method for extracting bacterial lipopolysaccharides. Take 200g of inactivated bacterial cells, add non pyrogenic double distilled water (PFW) and stir to form a bacterial suspension. Add equal volume of 90% (W/W) re distilled phenol (preheated to 68 ℃ and adjusted to pH 7-10), at which point the bacterial concentration in the mixture reaches 20ml per gram of bacterial cells. Stir the mixture in an ice bath for 30 minutes, then cool (continuously stir) the mixture in the ice bath to around 10 ℃. Centrifuge at 7300g and 10 ℃ for 1 hour, carefully remove the upper phase liquid, combine the middle and lower layers, measure the total volume, add PFW to the total volume of the original mixture, and repeat the extraction once more. Combine the two phases and add ethanol to a final concentration of 25% (V/V). At the same time, add NaAc (final concentration 10mmol/L) and CaCl2 (final concentration 1mmol/L) and let it stand overnight at 3-8 ℃. On the third day, centrifuge at 7300g for 1 hour, and collect the precipitate to obtain crude LPS. Suspend this crude extract in 200ml of 50mmol/L Tris HCl (pH 7.2) buffer, add it to a dialysis bag (Mwco=12000) and equilibrate it at 37 ℃ in 50mmol/L Tris HCl (pH 7.2) buffer. Then add 50mg RNase and 50mg DNase I to the dialysis bag and replace the dialysis solution. Stir the dialysis solution at 37 ℃ for 6 hours. Add 100mg of proteinase K to the dialysis bag and replace the dialysis solution. Stir the dialysis solution at 37 ℃ for 24 hours. Then dialyze in PFW at 4 ℃ for 2 days, changing the dialysate twice a day. Suck out the dialysate at 64000 g, centrifuge at 3-8 ℃ for 5 hours, discard the supernatant, suspend and precipitate with PFW, and then add an equal volume of 90% (V/V) re distilled phenol (preheated to 68 ℃ at pH 7.0) and re extract once (using the same method as described above). Carefully remove the supernatant obtained by centrifugation after extraction, and dialyze it in PFW at 3-8 ℃ for 2 days. Remove the dialysate and analyze its concentration by UV scanning in the wavelength range of 190-340nm. After vacuum freeze-drying, store at T-20 ℃ Preparation of LPS. Dilute LPS with sterile physiological saline to 16g/L, and a set of injection formula is: 13 μ l diluent+500 μ l PBS+500 μ l complete Freund's adjuvant; One group consists of 13 μ l diluent+400 μ l PBS+5 μ l pertussis concentrate.

　　(2) Animal sensitization: Healthy adult Wistar rats were sensitized by subcutaneous injection of 200g/kg liquid paraffin into the abdomen at a dose of 1ml per rat one week ago. Select individuals without abnormal reactions for LPS injection.

　　(3) Inject LPS into sensitized rats, with the first formulation of LPS used for foot pad injection, 0.4ml per rat, and the second formulation used for intraperitoneal injection, 0.15ml per rat.

　　2. Observation indicators and analysis

　　(1) Eye symptoms: LPS injection leads to severe dilation and congestion of iris blood vessels, as well as adhesion of the anterior chamber angle; Choroidal vascular hyperplasia with diffuse infiltration of blood cells, accompanied by increased white blood cells, local edema, and wrinkles; Retinal and choroidal detachment, local edema and hyperplasia, and proliferation of pigment epithelial cells in the retinal pigment layer; Local protein like exudation of the retina affects the adjacent ganglion cell layer, while the fibrous layer is obscured or tends to be absent by the exudate. The protein like exudate exhibits heterogeneity, with disordered arrangement of cells in the connected ganglion cell layer, and fibrosis occurring in the vascular wall and adjacent tissues; The fibers in the local fibrous layer cluster into bundles and are distributed throughout, with some experiencing severe fibrosis. At the same time, thrombus like substances can be seen forming in the blood vessels between them. A series of clinically visible inflammatory reactions, whose symptoms are consistent with uveitis. At 4 hours, dilation and congestion of iris blood vessels were observed; At 8 hours, corneal edema, anterior chamber opacity, Thydull sign (+), slight exudation, blood vessels still congested, iris edema worsened and expanded in scope; At 12 hours, the reaction reached its peak, with high swelling of the iris, severe dilation and congestion of blood vessels, visible exudation, anterior chamber hemorrhage, and visible flocculent floating in the anterior chamber. Thydral sign (+) and pupil constriction were observed; At 24 hours, the above symptoms were relieved.

　　(2) Pathological examination: corneal epithelial edema, loose arrangement of corneal stroma, increased and congested ciliary blood vessels with protein like exudation, severe dilation and congestion of iris blood vessels, and adhesion of anterior chamber angle; Choroidal vascular hyperplasia with diffuse infiltration of blood cells, accompanied by increased white blood cells, local edema, and wrinkles; Retinal and choroidal detachment, local edema and hyperplasia, and proliferation of pigment epithelial cells in the retinal pigment layer; Local protein like exudation of the retina affects the adjacent ganglion cell layer, while the fibrous layer is obscured or tends to be absent by the exudate. The protein like exudate exhibits heterogeneity, with disordered arrangement of cells in the connected ganglion cell layer, and fibrosis occurring in the vascular wall and adjacent tissues; The fibers in the local fibrous layer cluster into bundles, and some of them undergo severe fibrosis. At the same time, thrombus like substances can be seen forming in the blood vessels between them. In addition, the dilation and congestion of blood vessels in the retina can be clearly seen in the slices, especially the dilation of capillaries in the inner and outer layers, as well as a large number of fibers surrounding the retinal aortic wall in the optic disc. The tightly connected part of the retina shows hyperplasia or swelling, and the fibrous layer is fibrotic.