| Objective 1.Learn and master B16F10 melanoma cell culture method;2.Learn and master the experimental techniques of measuring cell proliferation and viability by MTT method,tyrosinase activity by dopamine oxidation method,melanin content by Na OH dissolution method and protein expression by Western blot;3.The effects of kaempferol on the proliferation,melanin synthesis and tyrosinase activity of B16F10 melanoma cells were detected;4.Make statistical analysis on the test data,find out the effective concentration of kaempferol to inhibit melanin synthesis and tyrosinase activity,conduct signal transduction experiment with the effective concentration,and find out the best concentration for formal signal transduction experiment;5.To detect whether kaempferol has an effect on melanin synthesis and tyrosinase activity of B16F10 melanoma cells through PI3 K / Akt signal transduction pathway.Methods 1.B16F10 melanoma cells in logarithmic culture period were divided into experimental group(kaempferol group),positive control group(arbutin group),negative control group(DMSO group)and blank control group(simple cell culture group).Kaempferol and arbutin at concentrations of 25,50,100,200,500,1000 and 2000μmol/l were intervened for 24 h,48h and 72 h respectively.1.1 MTT assay was used to determine the level of cell proliferation in each group;1.2 determine the content of melanin by Na OH dissolution method;1.3 tyrosinase activity was measured by dopamine oxidation method;1.4 the effective concentration of kaempferol inhibiting melanin synthesis and tyrosinase activity is obtained by statistical analysis of the data for the next pre experiment.2.The effective concentrations of kaempferol inhibiting melanogenesis and tyrosinase(25,50,100,200μmol/l)were obtained from 1.4.The specific inhibitor BIO(2μmol/l)of GSKβ and kaempferol were combined to intervene B16F10 melanoma cells as the experimental group,with kaempferol,BIO and blank group(DMSO group)as the control.According to the methods in 1.2 and 1.3,the melanin content and tyrosinase activity of each group were measured respectively,and the best intervention concentration of kaempferol was obtained by statistical analysis of the data(50,100μmol/l).3.Detection of signal transduction pathway by Western blot:B16F10 melanoma cells in logarithmic culture period were divided into experimental group(50μmol/l kaempferol+BIO,100μmol/l kaempferol+BIO),negative control group(50μmol/l kaempferol,100μmol/l kaempferol)and blank group.The expression level of p-GSKβ in experimental group and blank group was detected by western blot.Kaempferol intervention cells were used as the experimental group(50μmol/l kaempferol,100μmol/lkaempferol),the blank group was used as the control,and the expression level of p-Akt in each group was detected by Western blot.4.Statistical analysis: SPSS 26.0 statistical software,the collected experimental data are expressed as mean ± standard deviation.The results of multiple groups were compared by One-way ANOVA,SNK-q test for homogeneous variance and Game-Howell test for uneven variance.The test level was statistically significant(P<0.05).Results 1.Master B16F10 melanoma cell culture method and obtain melanoma cells with good function and sufficient quantity for Experiment 2.Master the technical methods and operation steps of experimental design,and draw useful conclusions.2.1Kaempferol promoted the proliferation of B16F10 melanoma cells at 24 h,25-100μmol/l,but there was no significant difference compared with DMSO group(P>0.05),Kaempferol greater than 200μmol/l could very significantly inhibit cell proliferation(P<0.01);At 48 h,kaempferol greater than 50μmol/l could significantly inhibit cell proliferation compared with DMSO group(100μmol/l P<0.05,other P<0.01);At 72 h,kaempferol greater than 100μmol/l could significantly inhibit cell proliferation(2000μmol/l P<0.01,other P<0.05).In general,kaempferol inhibited the proliferation of B16F10 melanoma cells in a dose-dependent and time-dependent manner,At 24 h,48h and 72 h,there was no obvious cytotoxicity at the concentrations of less than 2000μmol/l,less than 1000μmol/l and less than 500μmol/l,respectively.At 24 h,when ≤200μmol/l,the inhibitory effect of kaempferol was weaker than that of arbutin,and the difference was statistically significant(P<0.05);At 48 h,when ≤100μmol/l,the inhibitory effect of kaempferol was weaker than that of arbutin,and the difference was statistically significant at 25μmol/l and 50μmol/l(P<0.01,P<0.05);At 72 h,the inhibitory effect of kaempferol was weaker than that of arbutin at 25μmol/l and 50μmol/l,and the difference was statistically significant at 25μmol/l(P<0.01).2.2Kaempferol inhibited the melanin synthesis of B16F10 melanoma cells at 25-2000μmol/l,24 h,48h and 72 h,and showed an obvious dose effect and time effect relationship.Compared with DMSO group,the difference was statistically significant(P<0.01).At 24 h,48h,72 h and25-2000μmol/l,the inhibitory intensity of kaempferol at the same concentration on melanin synthesis was stronger than arbutin,and the difference was statistically significant(24h,≥500μmol/l;48h,25μmol/l、≥500μmol/l;72h,≤100μmol/l P<0.01,other P<0.05).Kaempferol inhibited the tyrosinase activity of B16F10 melanoma cells at25-2000μmol/l,24 h,48h and 72 h,and showed an obvious dose effect and time effect relationship.Compared with DMSO group,the difference was statistically significant(24h,≥500μmol/l;48h,≥200μmol/l;72h,≥200μmol/l P<0.01,other P<0.05);The inhibitory intensity of kaempferol at the same concentration on tyrosinase activity was stronger than that of arbutin at 24 h,48h,72 h and 25-2000μmol/l,except 48 h,50μmol/l;72h,25μmol/l,the difference was statistically significant(24h,50-200μmol/l;48h,25μmol/l;72h,50、500μmol/l P<0.05,other P<0.01).In conclusion,kaempferol can significantly inhibit melanin synthesis and tyrosinase activity compared with arbutin at 24 h,25-200μmol/l.2.3 The effective concentration of kaempferol was 25,50,100 and 200μmol/l,and the action time was 24 hours.The combined treatment of kaempferol and BIO could weaken the inhibition of tyrosinase activity and melanin content caused by kaempferol at the same concentration,and there was significant difference between the two groups(P<0.05).When kaempferol was 50μmol/l and100μmol/l,the tyrosinase activity and melanin content of B16F10 melanoma cells increased the most after combined treatment with BIO.After BIO treatment alone,compared with the blank control group,the tyrosinase activity and melanin content of cells increased,and the difference was statistically significant(P<0.05).2.4 The best concentration of kaempferol was 50 and 100μmol/l,and the action time was 24 h.Compared with the blank control group,the expression of p-Akt in total protein increased after kaempferol treatment with 50 and 100μmol/l,and the difference was statistically significant(P<0.01,P<0.01);Compared with the blank control group,the expression of p-GSKβ in total protein increased after kaempferol treatment with 50 and 100μmol/l,and the difference was statistically significant(P<0.01,P<0.05).Compared with kaempferol group,kaempferol + BIO group can significantly reduce the expression of p-GSKβ,When the concentration of kaempferol was 50μmol/l,the difference was statistically significant(P<0.05).Conclusion 1.B16F10 melanoma cells were successfully cultured and grew well with sufficient quantity,which fully met the experimental needs.2.Kaempferol inhibited the proliferation,tyrosinase activity and melanin synthesis of B16F10 melanoma cells in a dose-time-dependent manner,and had no obvious cytotoxicity.The inhibitory effect of kaempferol at low concentration was significantly weaker than that of arbutin,and the inhibitory effect was gradually significantly higher than that of arbutin with the increase of concentration.3.Kaempferol may inhibit melanin synthesis and tyrosinase activity of B16F10 melanoma cells through PI3 K / Akt signal transduction pathway. |
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