Protective Effect Of Gentian And Baicalein On The Oxidative Damage Of Hpea-like Cells Induced By HiPS

Objective:1.Perfect the culture technique of hiPSC-induced Hepa-like cells2.Establish the oxidative damage model ofhiPSC induced Hepa-like cells3.To explore the liver protective effect of hiPSC and Baicalein on the oxidative damage model of Hepa-like cells induced by the Chinese herbal medicineMethod:1.hiPSC induces differentiation into hepatocytes:Before differentiation,the IPs cells are cultured in the coating culture bottle after the substrate gum wrap,and the culture liquid is hiPSC medium.Initiation of differentiation:Using cytokines 4 steps to induce differentiation:the 1th stage is 8 days,hiPSC differentiated into endoderm cells,namely,adding 100 ng/ml Activin A and 10 ng/ml bone morphogenetic protein 4 and bone morphogenetic proteins4(BMP4),20 ng/ml fibroblast growth factor2(FGF2),and B27 Supplements Minus UnsulininRPMI1640;The 2nd Stage is 5 days,the differentiation of endoderm cells to hepatocyte-like cells,namely,adding 20 ng/ml BMP4,10 ng/ml FGF2,and B27 Supplements in RPMI1640;P1hase 3rd is 5 days,the proliferation of hepatocyte-like cells,the addition of 20 ng/ml hepatocyte growth factor(HGF),and B27 Supplements in RPMI1640;4th Stage is 5 days,the type of hepatocyte maturation,the addition of 20 ng/ml Oncostatin M into the HCM(omitting the EGF).The changes of cell morphology in the No.1 and 3,12,23 days of human iPSCs were observed by inverted fluorescence microscope,and whether the liver cells were morphological in human iPSCs-induced hepatocytes,and the iPSCs and hepatocyte markers were detected by confocal laser technique:GATA4,hnf-4a,AFP,ALB and P450 proteins were expressed to identify the whole induction process.2.Hepa-like cell oxidation model modeling method:Using LO-2 cells as a preliminary test to determine the dosage of H2O2 oxidative damage modeling,will be in the period of growth of the LO-2 cells digested with 7,000 per hole in 96-hole plate,After 24 hours of sticking to the wall,the H2O2 working fluids(100,200,300,400,500,600,700,800,900,1000 ?mol/1)were diluted into 10 groups with RPMI1640-FBS Medium,and the control group of 0 u mol/L,A total of 11 groups,each set of 4 complex holes.After 24h,MTT assay was used to measure the survival rate to calculate the IC50 dosage of H2O2.The content of superoxide dismutase(SOD),glutathione peroxidase(GSH)and Malondialdehyde(MDA)and the level of alanine aminotransferase(ALT)and Aspartate aminotransferase(AST)in cell supernatant were detected by adding this dosage to the Hepa-like cells which were successfully induced.3.Study on the protective effect of Chinese herbal monomer gentian saponins on hepa-like cell oxidative damage model induced by HOO2Pre-test was used to determine the effective range of LO-2.The Hepa-like cells were successfully induced,and the oxidation model was prepared by adding 300 ? mol/1 H2O2.After molding 24h,discard supernatant,add 4 concentrations of gentian glucoside solution,in which the control group does not make the mold without the gentian,set up the module without Gentian Saponin,to detect the oxidative damage model 24h of the therapeutic effect of gentian.To investigate the protective mechanism of gentian on liver by changes of serum ALT,AST,SODandMDA in liver protection related biomarkers.4.Effect of Chinese herbal monomer baicalein on liver protective effects of H2O2 induced Hepa-like cell oxidative damage modelPre-Test uses LO-2 to determine the effective range of Baicalein.The Hepa-like cells had been successfully induced,discarded supernatant,added Baicalein solution of different dosage concentrations,added Baicalein 2h,abandoned supernatant,added 300 ?mol/1 H2O2 to make oxidation model,and set up control group without mold without Baicalein,and set the module without Baicalein.And the cells were collected after 24h.The experiment was repeated three times to detect the preventive effect of baicalein.The Hepa-like cells were successfully induced,and the oxidation model was prepared by adding 300 ?mol/1 H2O2.After molding 24h,discard supernatant,add 4 concentrations of gentian saponin solution,in which the control group does not make the model without the Gentian,set up the module without Gentian Saponin,to detect the role of Baicalein in oxidative damage of Hepa-like cell model,and detect sod,MDA,ALT,ASTResult:1.The changes of cell morphology in the differentiation of Hepa-like cells induced by human iPSCs:visible in the first day of induction,the cells still maintain the form of iPS,both as a set of growth,small cells and round,dense growth,the edge of the group is obvious.On the eighth day of the first stage of the induction end,the visible cell group was dispersed,the cells were larger and not round,but resembled elliptical.At the 13th day of the second stage induction end,the cell colony margin was completely unable to be seen at 5 times,the cells were completely dispersed,and the cell gap was larger,the cell size was different,and the nucleus was small in 20 times of microscope.In the third phase,the 18th day of the end of the induction,5 times the visible cells are larger,20 times the mirror visible cell shape of the same,gap larger and similar size.On the 23rd day of the fourth stage,the cells were arranged neatly under 5 times the microscope,and the size and shape of the cells were basically the same with the morphological features of the normal liver cells,which were in the form of multiple angles,large nuclei and circle.Immunohistochemical staining results:In the 8th day of the induction process,endoderm labeled protein gata expression.On the 13th day of the induction process,the hnf-4 a expression of labeled protein marked the differentiation of cells in vitro into hepatocyte differentiation lineage.On the 18th day of the induction process,the marker protein AFP expression,which is another important indicator of liver progenitor cell,is usually expressed in the original hepatocytes.On the 23rd day of the induction process and P450 were expressed,indicating that the cells had secretory and detoxification functions.The results showed that the induction process of cell differentiation was accurate and the induced hepatocytes were mature.The expression of statistical marker protein and nuclear protein can be calculated in more than 85%in different visual field.2.The dose of H2O2-induced oxidative damage in LO-2 cells can be used in Hepa-like cells in oxidative damage modelling and can be manifested when Hepa-like cells exhibit oxidative damage in a variety of enzymes.3.The effect of gentian on H2O2 induced Hepa-like cell oxidative damage model can be reduced to different degrees of intracellular ALT,AST,in which ALT is reduced in concentration dependence,and AST dependence on concentration is not obvious.SOD has different degree of elevation,MDA has different degree of decrease,the dependence on concentration is also not obvious,it shows that Gentian has therapeutic effect on iPSC induced Hepa-like cell oxidative damage model.4.In the prevention of oxidative damage model of H2O2 induced hepatic-like cells,Baicalein has a different degree of ALT,AST and concentration dependence,which shows that the effect of Baicalein on the integrity of liver membrane structure is better.SOD has elevated in different degree and showed obvious concentration dependence,The MDA had different degrees of decrease,which indicated that Baicalein could improve the rate of liver cell scavenging free radicals.It is indicated that Baicalein has a preventive effect on iPSC induced Hepa-like cell oxidative damage model.Conclusion:1.the hiPSCs use four factor step inducing differentiation method may induce the human to induce the pluripotent stem cell to differentiate into the Hepa-like cell,the successful differentiation cell has the liver cell form and show to express the important hepatocyte protein.2.H2O2 can be used as inducer of oxidative damage model of Hepa-like cells.3.Gentian and Baicalein have therapeutic and preventive effects on H2O2 induced Hepa-like cell oxidative damage model,and its mechanism is to remove oxidative free radicals and protrect organize cell membrane from destruction.