| Background Cellular senescence is the biological process of age-related declining ability of cell proliferation and degeneration in physiological function, which plays an important role in organismal aging. Cultured human diploid cell is one of the safest cell matrix for vaccine production. For safety and quality concerns, only early generation cells within two thirds of the life cycler can be used in vaccine production. However, little is known about what cellular aging processes change in different generation diploid cells in vitro. Meanwhile, no effective senescence evaluation methods is set so far. Therefore, study on senescent characteristic change of human diploid cell is of great importance in safety assessment of cell matrix and further study on aging-related diseases.Objective To study change of different generations in human embryonic lung diploid cells (Walvax-2 cell line) at different levels including cell microstructure, ultrastructure, protein and enzyme change, and cell growth, and to screen out reliable biomarkers for senescent evaluation of human embryonic lung diploid cells.Methods1. Sixteen generations of the Walvax-2 cell line were detected at present study, including the 20th,25th,30th,35th to 45th,50th and 55th generation.2. Microstructure and cell growth of Walvax-2 cells was observed using inverted microscope.3. Ultrastructure of Walvax-2 cells was observed using transmission electron microscope.4. Expression of β-galactosidase of Walvax-2 cells was analyzed using SA-β-gal staining method.5. Telomerase activity of Walvax-2 cells was analyzed using TRAP-PCR method.6. Ability of cell proliferation of Walvax-2 cells was analyzed by cell growth curve.7. Cell cycle phases and apoptosis of Walvax-2 cells were analyzed using Fluorescence activating cell sorter (FACS).8. Expressions of p16 and BMI-1 in Walvax-2 cells were analyzed using Western blotting method.Results:1. Microstructure and cell growth of Walvax-2 cells:the growth of early population (from the 20th to the 45th generations) was good, cells were observed with typical spindle shape, compact monolayer, polarized arrangement. There was no vacuole in the cell and the membrane were clear. Few intracellular granule was observed. Since the 50th generation, the growth of the cells became slower, the cells became darker and smaller, the shape began deformed, edge of the cell was blurred. At the 55th generation, the cells arranged irregularly and detached, microstructure were vague, large quantity floating dead cells appeared. The cell could be subcultured till the 58th generation.2. Ultrastructure of Walvax-2 cells in different generations:in the early populations (from the 20th to the 43rd generation), cells were observed to have normal volume, clear cytoskeleton, evenly distributed membrane microvilli, rich ribosome, smooth nuclear membrane. Senescent phenomenon appeared from the 44th generation---aptosis body,vacuole and cytophagosome were observed, cells were observed with larger endoplasmic reticulum, decreased ribosome, increased cytoplasmic pigment, folded nuclear membrane, smaller nucleolus. Senescent phenomenon was obvious in late population(from the 50th to the 55th generations), in addition to greatly increased aptosis body,vacuole and cytophagosome, cells were observed with deformed shape, enlarged cell volume, broken cell membrane, broken microfilament and microtubule, broken endoplasmic reticulum and mitochondria, ambiguous nucleus.3.SA-β-gal staining test results:The rate of stained cells was increasing with generation. For the early population cells(the 20th generation), percentage of the positively stained cell was 13.42%, while it was 37.72% in the late population cells(the 55th generation). The difference in stain rate were statistical significant among different generations (P< 0.05).4. Telomerase activity of Walvax-2 cells in different generations:There was a decreasing trend in the telomerase activity from the 20th generation to the 55th generations, however, there was no significant difference (P>0.05).5. Ability of cell proliferation of Walvax-2 cells:Indicating by the growth curve, there were significant differences in the growth rate between early populations and late populations. Compared with the 20th generation, the growth rate was much slower in the 50th generation. Proliferative activity in the 20th and 30th generations were stronger than the 40th and 50th generations(p<0.05), and the activity in the 40th generation was activer than stronger than the 50th generations(p<0.05)6. Cell cycle phases and apoptosis of the Walvax-2 cells:there is no change in the percentage of the G0/G1 phase in different generations, however, percentage of the G2 /M phase and S phase were decreased with cell growing process, and the S phase cell was rare at the 55th population. The amount of cell debris was increase with generation, and its percentage is 36.83% at the 55th population. There were significant difference in the percentage of the G2/M phase, S phase and cell debris in different generations(P<0.05).7. Expressions of p16 and BMI-1:The results of western blotting indicated that both p16 and BMI-1 were not detected in the Walvax-2 cells.Conclusion:1. Cell morphological changes, SA-β-gal positive staining rate, percentage of the S phase, G2/M phase and cell debris, as well as the cell growth ability were significant associated with generation time of the Walvax-2 cell. These characteristics could be selected as biomarkers for senescent assessment of the Walvax-2 cell.2. Althrough the telomerase activity was trended to inversely associated with generation time, further study was needed to determin whether it’s suit for senescent assessment of the Walvax-2 cell.3. Because p16 protein and BMI-1 protein were not detected in the Walvax-2 cells, they might not suit for senescent assessment of the Walvax-2 cell. |
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