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Contact inhibition phenomenon has important physiological significance, with the body organs, tissue morphology, size, and number of control various types of cells have a close relationship. The body's tissues and organs grow to a certain extent will naturally stop growing, rather than unlimited growth; in vitro process, when cell division and proliferation to a certain extent, to touch each other, the cells stop dividing. Generally, contact inhibition is due to consumption of the number of cells increased mitogenic factor of the local area; other is itself when contacting cells secrete a number of cell proliferation inhibitory factor. However Nakatsuji Y and HM.Robert research shows that: Astrocytes contact inhibition caused not by soluble factors, but by astrocytes induced by mutual contact between the induced proliferation inhibition mechanism. Contact inhibition mechanisms currently unclear. In order to study the principles of contact inhibition, we astrocyte cells as models to study the possible mechanism of contact inhibition, and explores the possible role of CD81 in them. This study consists of three parts. In order to observe the first part of contact inhibition induced astrocyte what is the role of some of the cells themselves secrete cell proliferation inhibitory factor, or because cells caused by contact with each other, this study purified astrocytes divided into three groups In confluent cells in a low, semi-confluence, completely confluent contact inhibition of conditioned medium was added, and cultured 20h, the number of cells by MTT assay, flow cytometry to detect cell cycle phase cell indices and statistical analysis. Conditioned medium did not inhibit the proliferation of astrocytes, it also had no effect on cell cycle. The results show contact inhibition astrocytes not due to contact of astrocytes induced the release of soluble factors, but by astrocytes induced by mutual contact between the induced proliferation inhibition mechanism. And further observations will be in contact inhibition of astrocyte cell separation technology to reach out to observe whether the cells re-enter the proliferative state, and after merging are again to be suppressed. We will be in contact inhibition astrocytes isolated, cultured and then, when the cell is approaching low confluence, half and full confluence confluence added BrdU, cultured cells were collected after 6h, in conjunction with FITC-conjugated anti-BrdU antibody were incubated Then the cell cycle by flow cytometry and cell BrdU positive rate. The results showed that: in contact inhibition astrocytes isolated, and then cultured, and the semi-confluent cells in low confluence, the cell proliferation rate increased again. When the confluence of the contact state of all cells, the cell growth rate decreased significantly. Showed that: in contact inhibition to reach isolated astrocytes, cells resumed proliferation. The contact with each other again, and inhibited cell proliferation. CD81 molecules that antiproliferative target antigen recognized by the antibody (Target of the Antiproliferative Antibody-1, TAPA-1), which is a molecular weight of 26KD non-glycosylated membrane protein, is a dry four transmembrane protein (tetraspanin) superfamily membrane containing four transmembrane region and extracellular region 2, are widely distributed in various tissues and cell surface, can affect cell adhesion, activation, proliferation and differentiation, cell morphology is changed, co-suppression cell formation and other biological functions. Sullivan, who found that immature astrocytes do not express or low expression CD81, as mature astrocytes, CD81 expression gradually increased, gradually reach adult levels. CD81 on the cell proliferation of the role of regulation, and in different cells play a different role. Therefore, the second part of the anti-CD81 antibody in different states purified cultured astrocytes proliferation inhibition effect. The purified astrocytes divided into three groups in the cell at a low confluence, semi-confluence confluence completely anti-CD81 antibody was added (4μg/ml), cultured cells were harvested after 18 hours by MTT assay and flow cytometry anti-CD81 antibody was observed in different states of astrocytes and analyzed statistically. The results show that compared with the control group, anti-CD81 antibody in low confluence half confluent astrocytes was significantly inhibited, the inhibition rates were 15.56% and 34.44%, on the confluence of the state is fully astrocytes slightly inhibited cells, the inhibition rate was 9.62%. Therefore, anti-CD81 antibody in low confluence and semi-confluent astrocytes were significantly inhibited, has been in contact inhibition astrocytes only slight inhibition. To further investigate the anti-CD81 antibody on astrocyte inhibition effect relationship, and the third part of the study of the anti-CD81 antibody for different time after astrocyte proliferation inhibition effect. The purified astrocytes cultured for 12 hours after the addition of anti-CD81 antibody (4μg/ml), respectively, for 18 hours, 32 hours and 96 hours later by MTT assay astrocyte cell activity, inhibition rate was calculated; to flow cytometry cell cycle phase. MTT assay showed that compared with the control group, the role of anti-CD81 antibody for 18 hours after 32 hours and 96 hours were on astrocytes significantly inhibited, the inhibition rates were 11.17%, 13.17%, 27.49%. Flow cytometry results showed that the anti-CD81 antibody for 18 hours, S phase cells index compared with the control group, an increase of 16.09, mainly astrocytes were blocked in S phase; effect 32 hours later, G0/G1 phase cells exponential increase compared with the control group 3.08; 96 hours, the cells index increased by 5.87 compared with the control group, the cells in G0/G1 phase main residence, namely the role of anti-CD81 antibody at different times after astrocytes were right there significantly inhibited the inhibition increased with the increase over time. Early anti-CD81 antibody that astrocytes were arrested in S phase, with the extension of time, and gradually make the cells were arrested in G0/G1 phase. Conclusion: The astrocyte contact inhibition caused not by soluble factors, but by astrocytes induced mutual contact between proliferation inhibition induced mechanism. In contact inhibition to reach isolated astrocytes, cells resumed proliferation. The contact with each other again, and inhibited cell proliferation. Anti-CD81 antibodies in low confluence and semi-confluent astrocytes were significantly inhibited, has been in contact inhibition astrocytes only slight inhibition. Anti-CD81 antibody for different time after astrocytes were significantly inhibited, inhibition increased with the increase over time. Early anti-CD81 antibody that astrocytes were arrested in S phase, with the extension of time, and gradually make the cells were arrested in G0/G1 phase. CD81 contact inhibition in astrocytes play a certain role, which may be anti-CD81 antibody combined with membrane CD81 was launched astrocyte proliferation inhibition mechanism.
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