Exploration Of Restriction Point In Normal Asynchronous Human Peripheral Blood Lymphocytes

Objective:To evaluate the effect of synchronizing exponentially growing human peripheral blood lymphocytes (PBLs) and Molt-4cells to quiescent state by traditional serum starvation.Methods:Shifted the exponentially growing human peripheral blood lymphocytes and Molt-4cells to serum-free medium for24、48h. Then the cells were collected and detected apoptosis(Annexin-V/PI)、proliferation(Ki67) and expression of cyclin E.Results:As exponentially growing PBLs were cultured in serum-free medium for24、48h, the apoptosis increased from (32.27±2.90)%to (61.82±2.47)%; Ki67decreased from (67.20±8.54)%to (51.87±0.59)%.; CyclinE+cells in G1phase decreased from (63.35±4.99)%before shift to (46.04±1.84)%and (44.46%±2.97)%separately; cells in S phase increased from (12.43±1.42)%before shift to (16.73±0.96)%and (20.70±1.86)%separately; cells in G2/M phase increased from(1.49±0.98)%before shift to (5.18±1.00)%and (8.06±1.38)%发separately.Similar results were obtained in exponentially growing Molt-4cells when shifted to serum-free medium for24%48h. Conclusions:Shifted the exponentially growing human peripheral blood lymphocytes and Molt-4cells to serum-free medium for24、48h according to the traditional serum starvation methords, the apoptosis rate increased and proliferation rate decreased at the same time. However, the percentage of S phase cells was high to20%after48hours’ starvation, failing to be synchronized to quiescent state. So, we can draw the conclusion that the traditional serum starvation can not fully synchronize exponentially growing cells to quiescent state. Objective:To confirm the existance of restriction point in normal asynchronous human peripheral blood lymphocytes(PBLs), and provide strong evidence for restriction point location in G1not GO phase.Methods:The fresh human peripheral blood lymphocytes(PBLs) were stimulated in serum-containing medium with1%PHA added for12,18and24h. Some PBLs were then shifted to low-serum medium for a total of48h. Collected the cells and detected the expression of CyclinE by flow cytometer to analyze the percentage of positive cells in G1-cyclinE-、G1-cyclinE+、S and G2/M phase. The other PBLs were shifted to serum-free medium for a total of48h. Collected the cells and detected the expression of CyclinE by flow cytometer to analyze the percentage of positive cells in Gl-cyclinE-、Gl-cyclinE+、S and G2/M phase, P21expression in G0/G1phase, ki67expression and percentage of BrdU incorporation in PBLs.Results:1、After stimulated by serum-containing medium with1%PHA for0、12、18、24、48h, the percentage of S phase in fresh human peripheral blood lymphocytes increased, which means that quiecent cells went through cell cycle gradually. 2、When the PBLs were shifted to low-serum medium(1%FBS) at12,18and24h, the percentage of S phase cells was about13%to24%from the beginning, which is failed to undergo the release process of quiecent cells.3、There were no cells entry S phase when the PBLs were shifted to serum-free medium at12h to total of48h, although75%of G1phase cells expressing CyclinE. When shifted at18h, the percentage of cells expressing CyclinE in S phase and G2/M phase was about19%and2%seperately, comparing with that of23%and3.7%at24h.4、The positive expression of ki67was(7.83±1.36)%when the PBLs were shifted to serum-free medium at12h to total of48h, while it reached to about (21.89±2.72)%and (39.98±7.51)%when shifted at18h and24h seperately.5、There was (2.18±1.37)%BrdU incorporation when the PBLs were shifted to serum-free medium at12h. However, the percentage come to (5.23±0.53)%and (12.06±3.49)%when shifted at18h and24h seperately.6、The positive expression of P21in G0/G1phase was82.45±4.61)%when the PBLs were shifted to serum-free medium at12h, while it decreasd to (49.89±19.11)%and (41.09±15.55)%when shifted at18h and24h seperately.Conclusions:1、After stimulated by serum-containing medium with1%PHA for0、12、18、24、48h, the quiecent cells went through cell cycle gradually, which made them to be a more suitable candidate in restriction point research.2、When the PBLs were shifted to low-serum medium(1%FBS) at12,18and24h, the percentage of S phase cells was about13%to24%from the beginning, which is failed to undergo the release process of quiecent cells. So, this method can not achieve the purpose of exploring restriction point in normal asynchronous human peripheral blood lymphocytes.3、Our group convinced that the restriction point, locating in G1but not GO phase, do exist in normal asynchronous human peripheral blood lymphocytes for the first time. For the human peripheral blood lymphocytes that stimulated by1%PHA in vitro, the restriction point locates between12h and18h.