Study On Inhibition Effects Of Stichopus Japonicus Acidic Mucopolysaccharide On Hela Cells

Objective:To Investigate the effects and the possible mechanisms of SJAMP in human cervical cancer Hela cell line in vitro.Methods:Hela cells were cultured in vitro and treated with various concentrations of SJAMP. Its effects on the cells were evaluated by detecting follow indexes:1 The effect of SJAMP on cells proliferation was evaluated by MTT assay.2 Immunocytochemical stain was used to detect the expression of P53 protein.3 The Hoechst 33258 method was performed to determine cell apoptosis.4 DNA electrophoresis method was performed to determine apoptosis.5 Rhodamine 123 staining and flow cytometry analysis were designed to investigate the status of the mitochondrial membrane potential.6 Western Blot was used to detect apoptosis, and detect the apoptosis related change of expression of protein Bax and Bcl-2.Results:1 MTT assay indicated a significant dose-as well as time-dependent growth inhibition was observed in SJAMP-treated Hela cells.2 The immunocytochemical stain showed that different concentration of SJAMP decreased the abnormal expression of P53 gene (p<0.05), and indicate time-dependent effect.3 The cell apoptosis morphological changes was observed using Hoechst 33258 staining. The SJAMP-treated cells were fewer and smaller than control group. It illustrate that SJAMP could induce cell apoptosis of Hela cells.4 DNA Ladder was observed after SJAMP treatment by agarose gel electrophoresis. It indicated that SJAMP could induce cell apoptosis.5 flow cytometry detection showed that compared with control group, SJAMP obviously decreased the expression of mitochondrial membrane potential and the effect was manifest with concentration rising (p<0.05) and had a dose-dependent relationship.6 Western Blot analysis showed:compare with the control group, the expressions of Bcl-2 proteins were down-regulated, whereas the expression of Bax was up-regulated in a concentration-dependent manner from 0 to 6.4 mg/mL in Hela cells. There is marked ratio of gray value of Bcl-2 and Bax difference among each density (p<0.05). It indicated that apoptosis protein (Bcl-2 and Bax) can be regulated after SJAMP-tread.Conclusion:1 The results showed that SJAMP had a significant time-dependent (1-5 days) as well as dose-dependent (0.8-6.4 mg/mL) inhibition effect on proliferation of Hela cells. SJAMP could inhibit proliferation. Its mechanisms might be related to many factors, such as inhibiting the abnormal expression of P53 gene.2 SJAMP could induce apoptosis in vitro. Its mechanisms might be associated with the down-regulation of the mitochondrial membrane potential and the path of cytochrome C; Down-regulation of the Bcl-2 protein expression and up-regulation of the Bax protein expression may be involved in SJAMP-induced apoptosis of Hela cells. But the certain mechanisms of molecular biology remain to be investigated.