Expression Of Occludin And ZO-1 In Cochlea Of Guines Pigs

Background: Blood labyrinth barrier (BLB) ensures relative stabilization of the inner ear microenvironment, retains normal physiologic function of inner ear. The normal BLB relies on the stabilization of blood vessel, which is one of the foundation of normal sense of hearing function. It consists of the lateral wall stria vascularis, zona occludens between marginal cells and basal cells, junctional complexs of cochlear axis and the consecutive no-window micrangium endothelial cells in ligament of cochlea. Stria vascularis (StV) in the lateral wall of cochlea is an important part of the hymeno-lateral wall stria vascularis, which contains plentiful micrangium. StV is mainly consisted of three layer cells, they are marginal cell, intermediate cell and basal cell. The basal lamina of micrangium is very thin. There are tight junctions(TJs) between the near lumina of blood vessel endothelial cells and marginal cells, their endothelial cells are circled by perithelial cells. The fibrilla ecphyma of perithelial cells extends to endodermis and forms tight junctions,too. The etabolism of StV is the most prosperous part in cochlea, it can excrete K+ and absorb epilymph fluid electrolytes and produce endolymph fluid positive potential. K+ aggregation in endolymph fluid and K+ circulation in endolymph fluid and ectolymph fluid is the prerequisite of retaining the normal function of bristle cells, and the foundation of maintaining the cochlear function and internal environment stabilization. Recently, researchers both in home and abroad have done a lot of studies on the morphology and the physiological function of BLB. But little research has focused on the point of molecule view between morphology and physiological function in BLB.TJs are complexes which is consisted of multiple proteins, they have complicate structure with a multiple functions. TJs not only can block cell spaces and consititue the structure of barrier, but also relate to signal transduction of intra-cellular and cell growth and differentiation. TJs also have related with many kinds of diseases. With the development of the study, it shows that TJs include many other important functions. For example, TJs have especial function like valve, it can adjust the access between water, molecule and ion; TJs have a function that they can prevent protein and lipid to diffuse freely in summit and bottom of cytolemma, and this function can retain polarity of cells;the protein in TJs participate in signal transduction of intracellular, it can come and go between cytoblast and TJs, and it can integrate to characteristic transcription factor and participate in expression and control of gene. Many pathological ,physical and chemical factors can effect the expression of TJs protein and change it. TJs protein mainly includes transmembrane protein occludin, claudins, JAMs, zonula occludens protein family and cytoskeletal proteins. Occludin and ZO-1 have very important roles in TJs protein. Molecular weight of transmembrane protein occludin is 60 KDa, which includes cell outer ring and four transmembrane areas. N-terminal and carboxyl terminus are all in the endochylema. The first of the cell outer ring and carboxyl terminus has highly homology in different organism genera. Occludin take part in participate of TJs. Molecular weight of ZO-1 is 220～225kDa, and it is located in zona occludens of many kinds of endothelial cells and endothelial cells. ZO-1 has multiple binding sites in kytoplasm. Carboxyl terminus of occludin contains a area named coiled-coil. This area is the interactive sites of occludin and ZO-1.The interaction of occludin and ZO-1 can connect transmembrane protein and cystoskeleton, adjustments signal transduction both in intra-cell and ecto-cell, changes contractility of actin and influences assembly and function of TJs in cells, adjustments permeability of cells.The expression and distribution of tight junction protein occludin and ZO-1 is correlated closely with permeability of endotheliocyte. So in this study, we choose occludin and ZO-1 for study objects of TJs protein, discuss the expression of occludin and ZO-1 in inner ear of guinea pigs.Objective: In this study, we used immunostaining and western blot to investigate the expression of occludin and ZO-1 in cochlea of guinea pigs. We chose brain and lung as positive control. We investigated the expression and distribution of occludin and ZO-1 in cochlea of guinea pigs. It is an useful study that we can know TJs protein whether or not have adjustment to BLB's function, it whether or not have important relationship with inner ear disease, it whether or not can change normal hearing and medication in inner ear to interference TJs protein whether or not get dependable curative effect.Materials and methods:1. Materials 40 healthy, pigmented, adult guinea pigs, of both sexes with intact Preyer's reflex. Weight 250-350 g, provided by the fourth military medical university experimental animal center. Rabbit anti-occludin and rabbit anti-ZO-1 are all from Zymed, USA. Goat anti-rabbit is from Sigma,USA.β-actin is from Santa Cruz,USA. The another reagents are analytical reagent.2. Immunohistochemistry Guinea pigs were anesthetized by 10 g/L napental(40 mg/kg) and perfused by 40 g/L paraformaldehyde(PFA) and subsequently killed by decapitation. Then took out the acoustic vesicle, discissioed the acoustic cochlea and round window membrane(RWM). Perfused acoustic cochlea several times, and put the cochlea into 40 g/L paraformaldehyde, stored at 4℃, after 24 hours, decalcified them by 100 g/L EDTA for 14 days. Meanwhile, pallium and lung were taken and put into 40 g/L PFA, after 24 hours they were stored at ordinary temperature in 700 mL/L alcohol. Then paraffin imbedded and cut parallel sheet along cochlear axle. The slice thickness is 5μm, the three paraffin section were dealed with rabbit anti-ZO-1,rabbit anti-occludin, and negative control section, and then deparaffinaged and hydrated. After washed with PBS, added first antibody, including ZO-1 rabbit polyclonal antibody(1:100) and occludin rabbit polyclonal antibody(1:100), stored at 4℃overnight; then washed with PBS; added to goat-anti- rabbit second antibody, and incubated for 2 hours at 37℃. Then added to ABC compound, and incubated for 1 hours at 37℃, after that washed them with PBS; DAB colorated, washed by tap water, dehydrated, cleared, mountinged, and observed with light microscope. In negative control PBS was used to take the place of the first antibody, other steps were the same as above.3. Western blot analyis Guinea pigs were anesthetized by 10g/L napental(40mg/kg) and subsequently killed by decapitation, acoustic vesicle were taken, the lateral wall stria vascularis and spiral ligament were taken quickly. Meanwhile, brain tissues and lung tissues were taken. They were stored at ?80°C freezer. Protein abstraction: three kinds of tissues protein were added into solution liquid, which included 20mmol/L Tris, PH7.4, NaCl, NaF, sucrose, sodium pyrophosphate, complete, Mini, EDTA-free tablet, then homogenated in ice, centrifuged at 12000 g for 5 min at 4℃. The supernatant was obtained. Three kinds of tissues protein content were determined using coomassie brilliant blue assay reagent. Then the three tissues were loaded to SDS-PAGE gel electrophoresis hole by according to 5μg each track, and loaded constant pressure 120 V/ 40mA until bromchlorphenol blue reach to hemline of gelatum. They then were transferred to a blotting membrane at constant pressure 300mA. This samples were stained and all blots were blocked for 2 hours in 50 g/L milk powder with TBST buffer. Three tissues were separately added rabbit anti-ZO-1(1μg/mL),rabbit anti-occludin (2μg/mL)andβ-actin(1:200), and stored at 4°C overnight. After that the membrane was washed by TBST buffer 2 times, and 10 min for each time, then washed by TBS buffer 1 time for 10 minutes. Then second antibody labeled by HRP was added, and incubated for 60 minutes, the membrane was washed additional 3 times, 10 minutes each time. ECL chemiluminescence system detected protein expression level of ZO-1, occludin andβ-actin. We analyzed the molecular mass of positive samples and the molecular mass according to signal power. The proportionality of gray scale of aim sample andβ-actin sample represent protein expression level.4 Statistical analysis of data Statistical analyses were carried out with a commercial software program SPSS 13.0, the data was expressed by x±s, the data beyond two groups was tested using ANOVA variance analysis, P<0.05.Results:1. Immunostaining results of occludin, ZO-1It can be found in light microscope, that occludin and ZO-1 was stained for buffy positive particle in micrangium endotheliocytes, spiral prominence cells, external sulcus cells, margin cells and based cells of the lateral wall stria vascularis, bristle cells and supporting cells in organ of Corti。But there was no immunization signal in stria vascularis cells using PBS for first antibodies; in brain, there was strong positive staining around intra-wall of micrangium, obviously in great vessels. Besides, astrocyte cells also have been stained; in lung, most of the positive particle was expressed in intra-wall of micrangium endotheliocytes and alveolar epithelial cells.2. Western blot results of occludin, ZO-1Two protein expression in three tissue were identified using occludin and ZO-1 antibodies, they were detected as a positive band at 220KDa,60KDa,43KDa, when usingβ-actin for intra-control. Gray scale scanning software was used to analyze Gray scale, statistics analytic result shows that occludin and ZO-1 were expressed in all of the tissues. The protein expression of the occludin and ZO-1 in brain is less than that in cochlea 67.11%(P<0.05),46.82%(P<0.05). The protein expression of the occludin and ZO-1 in lung is less than that in cochlea 56.79%(P<0.05),60.44%(P<0.05). The protein expression of the occludin in brain is less than that in lung 31.39%(P<0.05). The protein expression of the ZO-1 in brain is more than that in lung 25.61%(P<0.05).Conclusion:After Immunostaining, we found that TJs protein occludin and ZO-1 was stained for buffy positive particle in cochlea of guinea pigs in light microscope. These strong expression are mainly distributed in the stria vascularis, spiral ligament, organ of Corti and so on. Western blot analysis showed that an expression of the occludin and ZO-1 in cochlea is higher than that in brain and lung. Transmembrane protein occludin and ZO-1 protein has very complicated and irreplaceable effect in TJs. Its structure and function is similar with BBB.