角膜炎症,大鼠模型 z-bo 2020-12-04 311

　　Background: The use of steroids in corneal inflammatory diseases is quite common. Long-term use of adrenal cortex hormones can produce ocular side effects such as glaucoma, cataracts and affect the corneal epithelium. Because of these problems, other anti-inflammatory drugs are being tested, such as the physiological serine protease inhibitor SLPI (secreted leukocyte protease inhibitor). These drugs inhibit the elastase activity of neutrophils (PMN) and also inhibit the nuclear translocation of the transcription factor NFKB, which blocks the activation of pro-inflammatory genes. Protease inhibition plays an important role in the control of tissue damage at inflammatory sites by proteases. A strict balance between protease and inhibition of wound healing and inflammatory diseases is essential. Imbalance leads to various inflammatory diseases such as breast hyperplasia, chronic bronchitis, and emphysema. SLPI is a low-molecular-weight protein that has been identified as a protease inhibitor secreted by the mucosa. In recent years, our laboratory has studied the role of SLPI in several inflammatory processes. SLPI treatment is not easy to maintain because of its short half-life in serum. In order to improve the biological activity of SLPI, our laboratory has modified it at the molecular level. Transglutaminase (TG) is composed of 9 enzymes that catalyze the formation of isopeptide bonds between free amines and acyl groups. These enzymes are involved in processes such as inflammation, re-epithelialization, neovascularization, and fibrous extracellular matrix synthesis. TG2 is located in the nucleus, cytoplasmic matrix and membrane compartment. TG2 is expressed at the inflammation site and activates phospholipase A2, NFkB and MAPK to stimulate inflammation. It is also involved in the pathophysiology of wound healing, pterygium, dry eye, and allergic conjunctivitis. Therefore, there is a strong hypothesis that inhibition of TG2 may be beneficial to the inflammatory process of the cornea at least at certain points. The N-terminal part of the protease inhibitor of the SLPI gene fusion is designated as pf-mc as a recombinant expression of the fusion protein. The fusion protein can anchor the expression of TG2 in different locations. It allows SLPI molecules to act locally and may increase the half-life of the protein. Corneal injuries cause very serious damage to the eyes. Cause inflammation and release collagenase and protease, severely damage corneal tissue. Inhibition of the NFKB pathway, activated by inflammatory cytokines, may reduce the amplitude of the inflammatory process and promote wound healing. For this purpose, our purpose is to evaluate the local effect of the above-mentioned fusion protein on a rat model of corneal alkali injury.

　　 Method: 36 male SD rats, 12 weeks old. These animals were screened without any eye disease. After general anesthesia with isoflurane, in the center of the right cornea, 1M NaOH alkali was used to damage 3 mm in 40 seconds. Then, the ocular surface was gently rinsed with 5 ml of saline. Animals were divided into three groups according to treatment methods: 1) pf-mc (200μg/ml); 2) SLPI factor (200μg/ml), 3) elution buffer. The treatment is topical application of 10 UL four times a day. The first administration was 30 minutes after the injury. Six animals in each group were sacrificed on days 3 and 7 of the disease.

　　PF-MC preparation: Purification and expression of pf-mc recombinant protein was prepared in BL21 E. coli. The bacterial particles are dissolved, sonicated, and centrifuged. Recovery of the soluble fraction and adding a column of Ni-NTA pf-mc protein. Add 2 ml of 50 mM phosphoric acid, 300 mM NaCl, 250 mM imidazole to the column to elute; pH=8. Reduce imidazole in protein samples Concentration, dialyzed with a final concentration of 2.5 mM phosphate buffer. Dialysis was performed overnight at 4°C, followed by protein recovery and aliquoting and dialysis buffer. The protein pf-mc was recovered by agar centrifugation. The quantification of protein concentration was analyzed with a microBCA kit.

　　 Evaluation of corneal epithelial repair time: Corneal re-epithelialization analysis was performed on the damaged cornea by fluorescent dye staining of the cornea. The epithelial defect retains the stained area, and the non-stained area completes the re-epithelialization process. Each group of six eyes was stained with fluorescein, and every 6 hours, under a cobalt blue microscope. 18 and 24 after the injury were taken with a digital camera. Use Adobe PS image processing software to standardize the pixels of the color area of the corneal size, and calculate the percentage of corneal ulcers.

　　 Evaluation of corneal turbidity: On the 7th day, the corneal turbidity was observed with a slit lamp microscope. Simply put, Grade 0: completely transparent cornea without a trace of turbidity. Level 0.5: Faint mist is detected by oblique illumination. Grade 1: Slightly cloudy but does not interfere with the visibility of the iris. Grade 2: More prominent turbidity and light iris obstruction. Level 3: Medium-density opaque, covering part of the iris. Level 4: Completely opaque and the anterior chamber structure is not visible.

　　 Histological examination: On the 3rd and 7th day, the animals were sacrificed. Take the eyeball and peel off the cornea. Then, 15 micron sectioning and HE staining were performed on the cornea. Microscopically examine the four parts of each cornea. The evaluation of the cornea includes the morphology and number of layers of corneal epithelial cells; interstitial cell count and identification of new blood vessels. The analysis was performed by two personnel. Analysis of neovascularization. According to the corneal diagram, the new blood vessels are classified into shallow, medium and deep from epithelium to endothelial cells. The expansion of new blood vessels is recorded according to the number of blood vessels in the cornea from the edge to the center of the microscopic field. Immunofluorescence analysis: The rats were euthanized 7 days after injury, the cornea was removed, fixed in 4% paraformaldehyde for 6 hours, and then immersed in 4 glucose solutions (5%, 7.5%, 10%, 20%) )overnight. Freeze with liquid nitrogen. The 15-micron-thick sections were incubated with anti-mouse vascular endothelial growth factor polyclonal antibody (VEGF) (1:500) at 4°C. Incubate with donkey anti-mouse immunoglobulin G receptor for 30 minutes. Use immunofluorescence microscope for evaluation.

　　Result: Evaluation of corneal epithelial repair time: 18 hours after injury, in the pf-mc treatment group, the first cornea was completely epithelialized. At 24h, 4 and 3 complete corneal epithelialization occurred in pf-mc and SLPI, respectively. Buffer-treated animals did not show complete re-epithelialization and all corneas were still incomplete. At 18 hours after injury, the area rate of corneal ulcers in the pf-mcp treatment group was significantly lower than that in the SLPI and buffer treatment groups.

　　 Corneal turbidity: After six hours of injury, fluorescent dyes show that 3 mm central epithelial cells stain the three groups of corneas after alkali burns. On the 7th day, the difference in corneal opacity scores between the three groups was statistically significant. The pf-mc treated eyes restore corneal transparency, and a clear anterior chamber structure can be seen through the cornea. However, not all animals in the buffer treatment group recovered corneal transparency.

　　 Corneal epithelium: Three days after injury, except for one animal in the pf-mc treatment group, the rest of the animals had three-layered epithelium. The degree of stratification is greater than other animal groups. Seven days after injury, histological analysis showed that the cornea of all animals in the pf-mc group was stratified at least three layers. The cell morphology of these animals looks quite normal. However, animals in other groups showed one or two stratifications of thin corneal epithelial cells. Corneal healing is unstable.

　　 Corneal stromal cell count: Three days after injury, the number of neutrophils and total cells in the pf-mc group decreased compared with the SLPI and buffer-treated animal group. Seven days after injury, the pf-mc treatment group had fewer cell counts in the corneal stroma than other treatment groups.

　　 Corneal neovascularization: 7 days after injury, animals in the pf-mc group had only superficial blood vessels, while blood vessels were found in the third cornea or even deeper in the other groups. Only the buffer treatment group found new blood vessels in the 6th zone or even deeper. Three days after injury, the pf-mc group rarely showed neovascularization, while the other groups had neovascularization.

　　 Conclusion: The SLPI transglutaminase fusion protein has good anti-inflammatory and anti-angiogenic properties for local treatment of rat corneal alkali burn model. This fusion protein is expected to be used for testing other eye diseases.