| Objective: Leukemia is one kind of malignant hematopoietic tumor which threatens the health of humanity. So far, traditional chemotherapy and radiotherapy are still the most significant methods. Reducing the adverse reactions and preventin of palindromia, organism immunity elevating etc. will be the emphasis of leukemia therapy. Ginseng radix is one of the famous Chinese traditional medicinal materials which have the positive influence to hematopoietic system. The most important active component of ginseng radix is ginsenoside which has little side effect and the effect of two-ways regulation. Respecting drug induced apoptosis usually need a large dose and have a not perfect effect, tumor cell senescence induced by natural drug active component has been the mainly orientation which a large number of scholars followed with. Ginsenoside Rg1 is to be adopted to human leukemia K562 cell line to induce its senescence, and senescence related index will be detected in our research, meanwhile, we will discuss the molecular mechanism, to the aim at providing evidence of tumor cell senescence biological theory and the effective target of natural drug component, significantly, That will provide original approach with feasibility in clinical therapy.Methods: K562 cell were cultured in conventional, when it growths in exponential phase, experiments were divided in two groups, control group and experimental group. Cells were play in 96-well plates, experimental group incubated for 24h, 48h and 72h with ginsenoside Rg1 at the concentration of 5μmol/L, 10μmol/L, 20μmol/L, 40μmol/L and 80μmol/L, each time spot and drug level were set four repetitions, control group were treat with out drugs and cultured in conventional. After 24h, 48h and 72h, K562 cells were enforced in MTT experiment to observation that whether ginsenoside Rg1 play roles in cell proliferation on K562 cell, and select optimization working concentration. Furthermore control group and ginsenoside Rg1 induction ageing experimental group(20μmol/L, 48h)were detected by flow cytometry to investigate cell cycle and cell cycle blockage phenomenons of cell senescence; Then observe control group and experimental group by SA-β-Gal staining and difference in positive staining analysis with statistics. We separate mononuclearcell from medulla ossium of normal mouse, set groups as before then it were induced by ginsenoside Rg1, studied the role of ginsenoside Rg1 to hematopoietic system normal cells; prepare cellogran cultivation system, play control group and experimental group K562 cell in it, appreciate reproductive activity by counting clones. We adopt Southern blotting to determine telomere length of K562 cell and observate their difference then judgement whether experimental group cell becom ageing by replicability ageing approach. RT-PCR and Western blotting were performed to investigate senescence related gene p16, p53, p21, Rb mRNA expression and senescence related protein expression of P16, P53, P21, Rb, this will help to determine K562 cells aging and its signal pathway; We use transmission electron microscope to studied ultramicro morphologic feature of control group and experimental group K562 cells to observation whether these cells have senescent character or not.Results: The results showed that ginsenoside Rg1 significantly inhibited K562 cell proliferation, the optimal concentration was 20μmol/L and 48h was the best reaction time; Compared with the control group, experimental group cells was significantly arrested at G2/M phase; SA-β-Gal staining results displayed that in the experimental group cells significantly changed older, the positive rate was significantly higher and the difference was statistically significant; In the experimental group the ability of cell colony formation was attenuated, the number of colony formation was statistically significant compared with the control group; the telomeres of cells were significantly shorter, showing characteristics of replicative senescence; the aging related gene and protein expression was significantly increased, the differences of OD values were statistically significant; The results of projection electron microscope showed that cells significantly changed older, such as cell augment, the agglutination and fragmentation of heterochromatin, the enlargement of mitochondrial volume and lysosomal volume, fragmentation of Golgi and so on.Conclusion: The cell proliferation of K562 can be suppressed by the ginsenoside Rg1 in a certain range of drug concentration. It may be the consequence of aging induced by ginsenoside Rg1. The results of cell cycle arrest, the positive staining rate of SA-β-Gal, Cloning culture experiment, Telomere length detection, supermicro-morphology observation have shown that the aging changes of K562 in experimental group. The detection of aging related gene and protein shows that the K562 cell was induced into aging by the ginsenoside Rg1 via the pathway of p53-p21-Rb and p16-Rb. |
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