The Study On Propofol Regulating Hippocampus Precursor Cells Proliferation And The Mechanism

Part 1 Effects of propofol on the proliferation of culturedhippocampus precursor cellsObjective To investigate a practical method to culture the neural precursor cells from hippocampus of neonatal Wistar rats, and to study the effects of propofol on its proliferation. Methods Hippocampus precursor cells obtained sterility from the new-born Wistar rats and cultured in vitro were identified with immunocytochemistry methods. Meanwhile, the second passage cells were cultured in 96-well plate. 24 hours late, the cells were treated by different drugs. The cells were grouped into the control group that there was no drug in the culture medium and the propofol group with three different concentration ( the ultima concentration was 0.5, 2.5, 12.5μmol·L^-1 respectively )(n=8). 72 hours after thecells treated by drugs, MTT method and ³H-TdR absorption were detected to determine the propofol effect on the precursor cells' proliferation. Results With the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) in the medium, the cells isolated from the newborn rats' hippocampus could continuously proliferate and form into cell clones (neurospheres). Furthermore, the neurospheres could express the neuroepithelial stem cell protein (nidogen), and bromodeoxyoridine (BrdU), which given exogenously, could be tracked in the neurospheres. Then remove EGF and bFGF from the culture medium, the precursor cells could be induced to differentiate. The differentiated cells could express NSE and GFAP. Contrasting to the intralipid group, the A_570nm value and the ³H-TdR absorption increased with the treatment of low concentration propofol (0.5, 2.5μmol·L^-1) ( P(2.5μmol·L^-1) vs LP: MTT method,A_570nm value 0.53± 0.03vs0.49± 0.03; ³H-TdR absorption, cpm value 7884±309 vs 4849±659 ) (P<0.05). Conclusion The cells obtained and cultured by this method process the property of the neural precursor cells. Low concentration of propofol can enhance the proliferation of hippocampus neural precursor cells. Part 2 The role of signal pathways during the precursor cellsproliferation promoted by PropofolObjective To study the role of the cAMP-PKA-CREB and the Ras-Raf-MEK-ERK-CREB signal pathways during the proliferation of hippocampus precursor cells promoted by propofol. Methods The method of cells culture was the same as described before. 1. The effects of H89 on the HPCs proliferation promoted by propofol: the second passage cells were cultured into 96-well plate for 24h. Then the cells were treated with different drugs to evaluate the proliferation ability. The cells were grouped as following, the control group (there was no drug in the culture medium), the H89 ( H89 was added into the culture medium, and the ultima concentration was 10μmol·L^-1) group, the H89 vehic control group (the concentration of vehic was the same as in the H89 group), the propofol ( propofol was added into culture medium, and the ultima concentration was 2.5μmol·L^-1) group, and the combination of propofol with H89 or H89 vehic group. 2. The effects of PD98059 on the HPCs proliferation promoted by propofol: the experiment was grouped the same as that of H89 with the H89 replace by PD98059. After the cells treated by drugs for 72h, the microscope was used to check the morphologic changes of precursor cells and CCK agent was used to check the ability of precursor cells proliferation. Results The ability of precursor cells proliferation decreased remarkably when treated with H89 or PD98059 (both of the concentrations were 10μmol·L^-1) . The CCKtest results show that both of them can counteract the ability of propofol facilitating cell proliferation (P<0.01). Conclusion The facilitating cell proliferation ability of propofol is related with the cAMP-PKA-CREB and Ras-Raf-MEK-ERK-CREB signal pathways. Part 3 The study on effects of propofol on corticosteroneinduced precursor cells proliferation inhibition.Objective To study the effects of propofol on the proliferation of cultured hippocampus precursor cells (HPCs) inhibited by corticosterone, which were isolated from new-born Wister rats' hippocampus, and the relationship with GABAa receptor. Methods l.The cell proliferation prohibited model was built by corticosterone with the culturing precursor cells isolated from new-born Wistar rats' hippocampus. 2. The HPCs were culture in 96-well plate for 24h when were used for experiment. The cells were divided into different geoups according to the drugs (6 holes per group). When the cells treated by different drugs for 72h, A_570nm value and ³H-TdR absorption were detected to evaluate the propofol and bicuculline's effects on the proliferation. 3. The HPCs cultured in 6-well plate were used to qualify the expression of GABA_A receptor by immunocytocheminal method. The HPCs cultured in 96-well plate treated by different drugs for 4h were used to quantify the changes of GABAa receptors on cells membrane by cell-ELISA. 4. The HPCs were cultured in 35mm culture capsules. After treated by different drugs for 4h, the cells were collected to study the changes of the GABAa receptor expression on the cells membrane by the method of western blot. Results The proliferation of HPCs can be inhibited by corticosterone according to the concentration and the concentration decided for this experiment was 100μmol·L^-1. The proliferation of the HPCs inhibited by the corticosterone was improved by Propofol (0.5, 2.5μmol·L^-1) (P<0.05). However, the effects ofpropofol facilitating HPCs proliferation could almost counteract by the Bicuculline. HPCs membrane expressed GABAa receptors. GABA_A receptors expressed on HPCs membrane decreased significantly when the cells were cultured with corticosterone for 4hr, however GABAa receptors expression increased when cultured with propofol (P<0.05). And propofol could reverse the effects of corticosterone when the cells cultured with both of them. The western blot result showed that, contrasting to the control group, propofol could increase GABAa receptor expressing about 16% while corticosterone could decrease about 14%. Conclusion Propofol with the concentration from 0.5 to 2.5μmol·L^-1 can facilitate the proliferation of the precursor cells which inhibited by the corticosterone. HPCs membrane expresses the GABAa receptors. The effects that propofol can facilitate the proliferation and reverse the inhabitation effects of corticosterone may relate to the GABA_A receptor's function. Part 4 The study on the effects of propofol and corticosterone onthe delayed rectifying K⁺ currents of cultured HPCs membraneObjective To record the outward K⁺currents of the hippocampus precursor cells, and the effects of propofol and corticosterone on the K⁺ currents. Methods the method of cells culture is the same as described before. The cells were cultured in 35mm culture capsules, and were grouped into control group, corticosterone (100μmol·L^-1) group, propofol (2.5μmol·L^-1) group and the co-administer of these two drug. The current was recorded after the cell was treated with drugs for 24h. Membrace currents were recorded in whole-cell voltage-clamp mode with the EPC-9 patch-clamp amplifier. The whole-cell voltage-clamp holding potential was -70mV. The voltage step protocols were ranged from -60mV to +70mV in10mV increments, and the duration is 50ms. All the experiments were carried out at room-temperature. Results The round cells with the similar size were chosen to record current. As the documents described, on the membrane of the HPCs precursor, the inward Na⁺ current could not be record. Replacing the KC1 with CsCl in the pipette solution containing 4-AP and TEA (10μmol·L^-1, respectively), we found that the current recorded were mainly the outward delayed rectifying currents. Compared with control group, corticosterone inhibited the largest outward delayed rectify K⁺ current but propofol facilitates the delayed rectify K⁺ current (P<0.05). Meanwhile, propofol could reverse the inhibition effect of corticosterone. Conclusion The delayed rectify K⁺ currents augmented when the HPCs treated with propofol, but diminished when treated with corticosterone for 24h. The accommodation of delayed rectify K⁺ currents maybe one of the most important reason affecting the HPCs precursor cells proliferation.