| Background and objectiveHepatocellular carcinoma (HCC) is a common malignant tumor, which is high malignance, rapidly development, too late to diagnosed, difficult to treatment, and has high mortality rate. Its pathogenesis is still not entirely clear, and lack accurate methods for early diagnosis and effective targeted therapies. To clear the pathogenesis of HCC especially HBV-related has been one of the key problems in the field of research on prevention and treatment of liver cancer, and the target to explore early accurate diagnosis and effective treatment targeted drugs of liver has been studied for years at home and abroad. Nano material is a kind of grain size of nano scale (10-9 m) of ultrafine material, has a unique structure, and the outer surface can adsor various molecules through non covalent force. It can also be combined with various chemical groups, the interior space can be entrapped ions and small molecules and can across the cell membrane. Therefore it has good prospect of application in biomedicine, including drug delivery, gene therapy and other aspects. Through the experiments of adding different concentration of titanium dioxide nanoparticles(Ti02 nanoparticles, TiO2-NPs) in cultured mediums, to study the effects of different concentrations of TiO2-NPs on biology function of hepatocellular carcinoma cells. Research of Sandra Vranic etc has showed that cells can uptake of NPS by international pinocytosis pathway that is energy dependent, at the same time, part of the NP can enter cells by passive diffusion. To further explore the transmembrane mechanism of TiO2-NPs, by adding different concentration of potassium ions in the culture medium, the influence of the cell membrane permeability maybe can changes the effect of TiO2-NPs on biology function of hepatocellular carcinoma cells, further to investigate the synergistic effect of potassium ions on TiO2-NPs.1 To investigate the mechanism of TiO2-NPs effect on human normal liver cell line L02 and human hepatic cancer cell line HepG2 cell biology function and its regulation.2. To study the mechanism of potassium ion effects on L02 and HepG2 cell biology function and its regulation.3 To investigate the synergistic effect of potassium ions on TiO2-NPs.Method1 the diameter and energy spectrum element analysis of TiO2-NPs by scanning electron microscopy;2 by cell counting, CCK-8 assay, flow cytometry and other methods, to study the affect of TiO2-NPs on L02 and HepG2 cell proliferation, cycle,and apoptosis.3. Using western blot, caspase3/7 detection reagent box to detect the Effect of TiO2-NPs on the expression level of L02 and HepG2 cell apoptosis associated protein caspase-3, membrane channel protein aENaC and the activity of caspase-3/7.4. Using western blot, JC-10 mitochondrial membrane potential detection Kit and ATP/ADP ratio assay Kit to detect the effect of TiO2-NPs on the expression level of mitochondria related protein SIRT3, VDACl,and ACSS1, mitochondrial membrane potential and the ATP/ADP ratio.5 By cell counting, CCK-8 assay, flow cytometry and other methods, to study the affect of potassium ion on L02 and HepG2 cell proliferation, cycle. and apoptosis,6. Using western blot, caspase3/7 detection reagent box to detect the Effect of potassium ion on the expression level of cell apoptosis-related protein Bax, Bcl-2, caspase-3, channel protein HERG and the activity of caspase-3/7.7. Using western blot and JC-10 mitochondrial membrane potential detection Kit to detect the effect of TiO2-NPs on the expression level of mitochondria related protein VDAC1, ACSS1, and mitochondrial membrane potential.Result1. The particle diameter of TiO2-NPs is about 15nm, the purity is about 95%.TiO2-NPs could inhibited the growth and proliferation of HepG2 cells, inhibited the S phase of cell cycle, and induced the apoptosis of HepG2 in concentration dependent, the inhibitory effect on proliferation and cell cycle in the S phrase is more obvious, the apoptosis rate was obviously increased in concentration dependent. While had no significant effects on L02 cells growth, proliferation, apoptosis, and cycle.2. Western blot results showed that, in HepG2 cells, TiO2NPs upradulated the expression of apoptosis-related proteins caspase-3, membrane channel protein aENaC in concentration dependent. In L02 cells, no significant difference of expression level changes of caspase-3 and aENaC.3. TiO2-NPs can make the mitochondrial membrane potential depolarized in HepG2 cell. And it can upgradulated the expression level of mitochondria related protein SIRT3 and VDAC1, and downradulated the expression level of mictochondria respiratory chain key protein ACSS1 in HepG2 cell. For L02 cell, the expression of SIRT3, VDAC1, ACSS1 had no obvious change.4. Potassium ions can inhibited the growth and proliferation of L02 and HepG2 cells, inhibited the S phase of cell cycle, and induced its apoptosis. As the concentration increased, the inhibitory effect on proliferation, cell cycle is more obvious, the apoptosis rate was obviously increased, showed significant concentration dependent,and the trends were more obvious in HepG2 cell.5. Western blot showed, potassium ion can upregulated the expression of apoptosis related proteins Bax, caspase-3 of L02 and HepG2 cells, and downregulated the expression of Bcl-2 significantly, by a concentration dependent manner. The trends were more obvious in HepG2 cell.6. Potassium ions could make the mitochondrial membrane potential depolarized,.and upgradulated the expression level of mitochondria pore protein VDAC1 and ion channel protein HERG in HepG2 and L02 cells, more significant in HepG2 cell. For HepG2 cell, potassium ions could downradulated the expression level of respiratory chain key protein ACSS1.ConclusionTiO2NPs induced the apoptosis, inhibited the growth proliferation effect and arrested the S phase of cell cycle of HepG2 cell significantly.Having no significant affect on the biological functions of L02 cell. TiO2-NPs TiO2-NPs induced HepG2 cell apoptosis by regulating the intracellular osmotic pressure, upregulated the expression of channel protein αENaC, mitochondrial porin VDAC1, depolarizing the mitochondrial membrane potential of HepG2 cells, resulting in the loss of Cyt-c and ATP, and further activate the caspase-3.Potassium ion inhibited the growth and proliferation, induced apoptosis of of L02 and HepG2 cells. Apoptosis induced by potassium ions may through the regulation of Bcl-2 family members and Caspase-3, depolarizing mitochondrial membrane potential of the L02 and HepG2 cell, especially in HepG2 cells.The affect of potassium ions on the cells of these biological functions could be related to ion channel protein HERG. |
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