The Primary Research On The Function Of CD81 In The Contact-inhibition Of Astrocyte

The phenomenon of contact-inhibition plays an important physiological meaning on controlling the shapes and sizes of organs and tissues and the numbers of all kinds of cells in the organism .when the tissues and organs arrive at some degree,they will stops to grow but not to unrestrictedly grow . In vitro assay, we also find that the cells will stop to divide when they divide and grow up some degree,arriving to col-contact.But the mechanism of action of contact inhibition is still unknown. In general, owing to the increasing number of cells dissipate divide factors, or the release or sequestration of soluble factors secreted at contact-inhibition. But the research of Nakatsuji Y and HM. Robert indicate:The inhibition of astrocyte proliferation did not appear to be mediated through the release or sequestration of soluble factors but rather could be induced by membrane associated factors. To examine the mechanisms mediating this inhibition of proliferation, we study the possible mechanisms and investigate CD81 play the possible function making the astrocyte as the model.The research has three parts. To examine the mechanisms that regulate the proliferation of astrocytes in the CNS were mediated through the release or sequestration of soluble factors or were induced by membrane-associated factors ; Purified astrocytes from newborn rats cerebral cortex, which were at low confluent,half confluent and full confluent, were incubated with condition medium for 20h. The activity of astrocytes was tested by MTT. Basing on MTT results, we calculated the inhibition rate of astrocytes. The cell cycles of the astrocytes were measured by the flow cytometery. Indicated by the result, Purified astrocytes which were at different confluent administration of condition medium after incubation for 20h have not inhibited the proliferation of astrocytes and have no function on cell cycle in contrast to the control groups. These data suggest that the inhibition of astrocyte proliferation did not appear to be mediated through the release or sequestration of soluble factors but rather could be induced by membrane-associated factors.To examine if astrocytes replated which were at full confluent grow into cell cycle and if astrocytes arrived at full confluent again would be at inhibition condition. When astrocytes replated which were at full confluent arrived at low ,half and full confluent after 6 hr BrdU pulse, Incubating with rat anti-BrdU antibody,We calculate the levels of BrdU incorporation and the cell cycle by the flow cytometer.The results indicate:Dividing the astrocyte that had in the condition of contact inhibition into signal cell and reculturing, replated at lower density and their level of proliferation will raise up again;When the cells came to full confluent reagain,the level of proliferation will decend again.CD81, target of the antiproliferative antibody-1(TAPA-1), is a member of the tetraspanin family of integral membrane proteins which plays an important role in biolog. CD81, expressed generally in a lot kinds of cells, is a 26-kDa surface protein composed of four transmembrane (TM) and two extracellular (EC) domains. It has been shown to influence cell adhesion,migration,division and proliferation, to alter cell morphology, to participate in signal transduction and so on. Sullivan and so on found that at embryonic day 18, the levels of CD81 are low, as development continues, the amount of CD81 expressed in astrocytes increases and at postnatal day 14 the levels approach those of the adult. CD81 have the ability to modulate the division and proliferation of cells and the different function on different cells.The second part of the study investigated the function of CD81 on inhibition of astrocyte at different confluent condition proliferation. Purified astrocytes from newborn rats cerebral cortex, which were at low confluent,half confluent and full confluent, were incubated with anti-CD81 antibody of 4μg/ml concentration for 18h. The activity of astrocytes was tested by MTT. Basing on MTT results, we calculated the inhibition rate of astrocytes. The cell cycles of the astrocytes were measured by the flow cytometery. The corresponding data were analyzed with SPSS statistical software. The results indicated : Purified astrocytes which were at low confluent,half confluent administration of anti-CD81 after incubation for 18h have remarkably inhibited the proliferation of astrocytes in contrast to the control groups . Inhibition rates of astrocytes reached 15.56% at at low confluent,34.44% at half confluent, 9.62% at low confluent, respectively. These data suggest that anti-CD81 antibody inhibits remarkably the proliferation of astrocytes at low confluent and half confluent, while inhibitis slightly the proliferation of astrocytes at full confluent, after incubation for 18h.To further investigate the time-effect function of CD81 on inhibition of astrocyte proliferation,Purified astrocytes from newborn rats cerebral cortex, which were cultured for 12h, were incubated with anti-CD81 antibody of 4μg/ml concentration for 18h,32h,96h. The activity of astrocytes was tested by MTT. Basing on MTT results, we calculated the inhibition rate of astrocytes. The cell cycles of the astrocytes were measured by the flow cytometery. The corresponding data were analyzed with SPSS statistical software. Indicated by MTT , administration of anti-CD81 after incubation for 18h,32h,96h have remarkably inhibited the proliferation of astrocytes in contrast to the control groups . Inhibition rates of astrocytes reached 11.17% at 18h, 13.17% at 32h, 27.49% at 96h respectively. By cell cycle analysis, the cells are arrested in S phase with the index of cells in S phase increasing 16.09 after incubation with anti-CD81 antibody for 18h, and the cells are arrested in G0/G1 phase with the index of cells in G0/G1 phase increasing 3.08 at 32h ,5.07 at 96h respectively. These data suggest that anti-CD81 antibody inhibits remarkably the proliferation of astrocytes after incubation for different time and the effect appeared time-dependent pattern. The cells are arrested in S phase at 18h and have been arrested in G0/G1 phase as time goes on.Conclusions: the inhibition of astrocyte proliferation did not appear to be mediated through the release or sequestration of soluble factors but rather could be induced by membrane-associated factors. Dividing the astrocyte that had in the condition of contact inhibition into signal cell and reculturing, replated at lower density and their level of proliferation will raise up again;When the cells came to full confluent reagain,the level of proliferation will decend again.The anti-CD81 antibody inhibits remarkably the proliferation of astrocytes at low confluent and half confluent, while inhibitis slightly the proliferation of astrocytes at full confluent, after incubation for 18h.The anti-CD81 antibody inhibits remarkably the proliferation of astrocytes after incubation for different time and the effect appeared time-dependent pattern. The cells are arrested in S phase at 18h and have been arrested in G0/G1 phase as time goes on.CD81 plays an important role on the contact-inhibition of astrocyte,the mechanism may be anti-CD81 body conbinated with CD81 initiating the mechanism of inhibition of astrocyte proliferation.