Study On Apoptosis Of Human Hepatoma HepG2 Cells Induced By Ganoderma Lucidum Peptides

In order to research apoptosis of human hepatoma HepG2 cells induced by Ganoderma lucidum peptides（GLP） in vitro and its mechanism,morphology of cells was observed under light microscope,fluorescence microscope and transmission electron microscopy respectively.Anti-proliferation effects of GLP were detected with methyl thiazolyl tetrazolium（MTT） colorimetric assay.The changes of Ca²⁺ concentration were examined by laser scanning confocal microscopy.Distribution of cell cycle and the rate of apoptotic cell were analysed by flow cytometry.The expression of apoptosis relevant gene was detected by western bloting,and the detection of Caspase-3 activity by colorimetric assay. The primary structure of GLP was identified by HPLC-MS/MS.Results are as follows:1.Morphologic observation of human hepatoma HepG2 cellsThe typical apoptosis features were observed,including chromatin condensation and massive on the nuclear edge,plasma membrane blebbing,cell shrinkage,nuclear condensation and formation of apoptotic bodies.2.Effect of GLP on proliferation,cell cycle and the apoptotic rates of human hepatoma HepG2 cellsMTT showed that GLP had anti-proliferation on human hepatoma ceils,the inhibitory effects were obvious,in a dose and time dependent manner.After treatment with GLP for 48h,GLP could cause G0/G1 cell cycle arrest in HepG2 cells,the cell proportion of G0/G1 phase was increased with the increase of GLP concentration.The apoptosis rate of early stage,late stage and total rate of apoptosis also increased with the increase of GLP concentration in a dose dependent manner.3.Apoptotic mechanisms of human hepatoma HepG2 cells induced by GLPIt was found that GLP caused an increase of intracellular Ca²⁺ concentration after HepG2 cells treated with GLP for 48h by LSCM.The result of western bloting showed that the expression levels of bcl-2 and Survivin,those were the apoptosis inhibiting gene, were downregulated and p53 which was the apoptosis improving gene was upregulated in a dose dependent manner.It was determined that Caspase-3 activity was activated by colorimetric assay,and there also was in a dose-dependent manner between GLP concentration and Caspase-3 activity.4.The primary structure of GLPThe amino acid sequences of two kinds of GLP were analyzed.When m/z was 566.8, the possible primary structure was as below:His-Ala-Leu（Ile）-Leu（Ile）-Leu（Ile） or Ala-His-Leu（Ile）-Leu（Ile）-Leu（Ile） When m/z was 814.5,the primary structure was as below:Leu（Ile）-Leu（Ile）-Leu（Ile）-Thr-Phe-His-Ala...