Expression Of CD81 In Normal, Detached And Reattached Rat Retina

CD81 is a member of tetraspanins (tetramembrane spanning) superfamily, and appears to be involved in the regulation of mitotic activity and the stabilization of cellular contacts. As a recently defined protein with unclear mechanism, it has caught increasing attention during these years. The present study investigates the distribution of this protein in normal rat retina, evaluates the effect of anti-CD81 antibody on the growth of retinal glial cells (RG) and retinal pigment epithelium (RPE) in vitro, and detects the expression of CDS 1 in detached and reattached rat retina.The purpose of the first part in this research is to observe the distribution of CD81 in the normal retina. By immunohistochemical analysis with an anti-CD81 monoclonal antibody EAT2 in the level of tissue, CD81 was found to express in the normal rat retina with a distinct laminar pattern of labeling. The positive staining existed throughout the ganglion cell layer, inner plexiform layer and outer plexiform layer in a dense reticular pattern, while a little on the membrane of RPE. By western blot analysis of sensory retina layer and RPE layer respectively with an anti-CD81 polyclonal antibody H121, CD81 was further confirmed to express in the both layers of normal rat retina. In cell culture, CD81 expression was located on the surface of RG and RPE and concentrated at regions of cell-cell contact.The second part is to find out the effect of CD81 on the growth of RGand RPE. The anti-CD81 antibody (EAT2 and H121) was added to cultured RG and RPE. After 7 days, MTT method was applied to examine the growth of these cells. It was found that the number of viable cells in the treated group decreased significantly. The most remarkable growth inhibitory effect of H121 on RG was 50.37%, while that of EAT2 on RPE was 72.7%. The potential of growth inhibition was related to the type and concentration of these antibodies.In the third part, we intend to examine the change of CDS 1 expression in the detached and reattached retina. An animal model of retinal detachment and reattachment was established by injecting 0.7% Healon into subretinal space, and extruding it from an incision of sclera after 7 days. Expression of CD81 was evaluated by immunohistochemical stain and flow cytometry. CD81 up-regulated in the detached sensory retina and RPE layer, which tended to peak at 7 days after detachment, and approached to normal till the 28th day. The proliferation index of retina was also measured by flow cytometry. Both the sensory retina and RPE layers showed elevation of proliferation index after detachment, with a similar time tendency to their expression of CD81. So far as reattached retina is concerned, the quantity of CD81 expressed in both layers turned down comparing to corresponding detached group. However, the downregulation of CDS 1 expression involved in the reattachment showed a delay to that of the proliferation index.In conclusion, our study demonstrates that CD81 could be expressed in the normal rat retina, especially on the membrane of RG and RPE. The combination of this protein with its antibody shows close relationship with the growth control of RG and RPE in vitro. The study also displays thatCD81 up-regulates during the retinal detachment, which tends to normal with the long-term of detachment or by reattachment. These data suggest that CD81 might play a role in the modulation of proliferative response of RG and RPE, probably through contact inhibition mechanism.