The Inhibition Effect Of Anti-CD81 On The Proliferation In Astrocytes

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 in all cells except erythrocyte,hemoblast and nerve, 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. The cultured astrocytes kept good abilities of division and proliferation. The proliferation of astrocytes is regulated by many cytokines and growth factors. The literature reported that the expression of CD81 is dramatically upregulated at the time of glial birth and maturation. 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. Our study also found that the levels of CD81 are low in low density of astrocytes, as development continues, the amount of CD81 expressed in astrocytes increases and at contact inhibition the levels approach the most. This implies that CD81 can mediate division and proliferation of astrocytes.In the first part, we observed the growth of astrocytes in an long time in order to investigate the growth characterization of the purified astrocytes in vitro, which the next study will base on. We divided the purified astrocytes from cerebral cortex of newborn rats into 14 hour groups. After incubation for different hours(6h,12h,18h,24h,30h,36h,42h,48h,54h,60h,72h,84h,96h,120h), we observed the proliferation by cell count and drew the growth curve of astrocytes, and we observed the cell cycle by the flow cytometer and drew the proliferation index curve of astrocytes The corresponding data were analyzed with SPSS statistical software. Results:①The growth curve of astrocytes were divided into three phases. The first phase continues from 0 to 24 hours, the slope rate is 0.64. The second phase continues from 24 to 84 hours, the slope rate is 2.69. At the first part of the phase(from 24 to 48 hours) the slope rate is 3.94 and the growth of cells is the fastest in the total process. The cells start the third phase after 84 hours, the slope rate is -0.005.②The proliferation index of astrocytes can reflect the proliferation of astrocytes in the first cell cycle. After the first cell cycle, the proliferation index reduces but the number of the cells is increase. The experiments above demonstrate : The growth process of purified astrocytes can be divided into 3 phases: latent phase(from 0 to 24 hours),logarithmic growth phase(from 24 to 48 hours) and stagnate phase(from 84 to 120 hours). By using merely the indexs, such as the proliferation index, it is circumscribed to estimate the proliferation in astrocytesIn the two part, we added anti-CD81 to the culture medium after cells were incubated at 37°C for 24 h and investigate the effect of anti-CD81 (antibodys against CD81) on the proliferation in astrocytes. Methods: Purified astrocytes from newborn rats,cerebral cortex were divided into 6 groups according to which applied different concentration (0,0.1,0.5,1,5,10μg/ml) of anti-CD81. The activity of astrocytes was tested by MTT. Basing on MTT, we chose 3 significative groups which applied different concentration (0,0.5,5μg/ml) of anti-CD81. After administration for 24h, the cell cycle of the astrocytes was measured by the flow cytometer. The corresponding data were analyzed with SPSS statistical software. Results:①By MTT , we found that the average optical density(AOD) values of astrocytes were reduced after administration with different concentration of anti-CD81 for 24h, that is , the number of astrocytes was reduced. That indicated anti-CD81 inhibited the proliferation of astrocytes and the effect appeared dose-dependent pattern.②By cell cycle analysis, we found that there was a progressive dose-dependent decrease in the index of cells in G0/G1 phase and increase in the index of cells in S phase. Such as, the index of cells in G0/G1 phase, is 82.73 in 0μg/ml, is 82.16 in 0.5μg/ml, is 78.58 in 5μg/ml. The experiments above demonstrate that anti-CD81 inhibites the proliferation of astrocytes and the number of astrocytes is reduced. Further, the index of cells in G0/G1 phase is decrease and the index of cells in S phase is increase after administration with anti-CD81. This study shows that anti-CD81 don't restrain the cells from G1 phase to S phase but the cells are arrested in S phase.Conclusion:①The growth process of purified astrocytes can be divided into 3 phases: latent phase(from 0 to 24 hours),logarithmic growth phase(from 24 to 48 hours) and stagnate phase(from 84 to 120 hours).②By using merely the indexs, such as the proliferation index, it is circumscribed to estimate the proliferation of astrocytes.③The proliferation of astrocytes which applied different concentration of anti-CD81 is inhibited and the cells are arrested in S phase of the cell cycle.