| Now,the pathogenesis of chronic sinusitis and nasal polyp disease are not complete known. Recently,as the developpment of a lot of experimental skills.more and more study approve that Nitric oxide(NO) and inducible isoform nitric oxide synthase(iNOS )play an important role in the pathogenesis and developpment of chronic sinusitis and nasal polyp disease.NO takes part in regulations of circulatory system,respiratory system,digestive system and nervous system.NO is a very small airy molecule.lt play an important role in inflammation and immune reponse.NO is synthetised by nitric oxide synthase.There are three type of nitric oxide synthase(NOS):neuronal nitric oxide synthase(nNOS), inducible isoform nitric oxide synthase(iNOS ) and endothelial nitric oxide synthase(eNOS ). nNOS and eNOS that are called structural nitric oxide synthase(cNOS) mainly regulate physiologyicalfuction.However, iNOS is produced in pathological process and play an important role in the formation and pathology of disease. iNOS exists in a lot of cells.It can be activated by mediators of inflammation and produces a lot of NO.NO can injure cells,increase permeability of blood vessel,inhibite platelet's adhere and gather and increase tissue edema.In the present study,we examine the level and distribution of iNOS mRNA in I and II typeof chronic sinusitis, nasal polyp disease and normal nasal membrane in situ hybridization.And we discuss the affection and clinical significance of NO and iNOS in the chronic sinusitis and nasal polypdisease.Materials and methods1 .Materials29 samples were obtained from 29 patients who received treatment in the First affiliated Hospital of Zhejiang University between September 2004 and Janurary 2005. We examined the expression of iNOS in human nasal tissues from patients undergoing I type of chronic sinusitis(n=8), II type of chronic sinusitis(n=8). nasal polyp disease(n=8) and normal nasal mucous membrane (n=5) .We cut the samples into 0.5 centimeter extent, embed the samples with wax and examined thesamples in situ hybridization. 2.Methods (1)When we taked the samples from the patients,we immediately used 4 percent ofparaformaldehyde to fix the samples for one hour.(2)We took away water,bathed wax and embed the samples.Then we cut the samples into sections which were 8 millimeter thick.(3)Through deparaffinization,the slides were brought to the liquid which was composed of 30 percent of hydrogen peroxide and disrillational water for 10 minutes in normal temperature.Then,the slides were rinced three times with disrillational water.The slides were rinced for 5minutes at every time.(4)Disclosure of mRNA piece:We added the pepsin which was watered by 3 percent of citric acid anhydrate in the slides.The slides were incubated at 37 °C for 30 minutes.Then the slides were rinced three times with phosphate buffered saline (PBS ) and once with disrillational water. The slides were rinced for 5minutes at every time.(5)The second fix:We added 1 percent of paraformaldehyde in the slides in normal temperature for lOminutes. Then,the slides were rinced three times with disrillational water.The slides were rinced for 5minutes at every time.(6)Pre-hybridization:We adde 20 μ　 t pre-hybridizing solution in every slides.Then the slides were incubated at 37°C for four hour.(7) Hybridization: We adde 20 μ t hybridizing solution in every slides.Then we put the sepecial cover glass on the slides. The slides were incubated at 42 °C overnight.(8) We took away the cover glass from the slides. Then the slides were rincedthree times with PBS. The slides were rinced for 15minutes at every time.(9) Adding of closed solution: The slides were incubated at 37°C for 30 minutes.(10) Adding of biotiny-digoxin: The slides were incubated at 37°C for 90 minutes. Then the slides were rinced four times with PBS. The slides were rinced for 5minutes at every time.(11) Adding of SABC: The slides were incubated at 37°C for 30 minutes. Then the slides were rinced three times with PBS. The slides were rinced for 5minutes at every time.(12) Adding of... |
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