| Objective In recent years,traditional Chinese medicine has shown superior advantages in tumor treatment.Traditional Chinese medicine has the characteristics of multiple targets and multiple effects.It has its own characteristics in improving the sensitivity of radiotherapy.Many Chinese medicines have improved the radiosensitivity of tumors.Report.This project is to start from the multiple tumors of liver cancer,observe the growth inhibition of matrine on human hepatoma Hep G2 cells and the apoptosis of liver cancer cells and the expression of MUC-1 protein in cells.Change and explore the mechanisms of change.Methods In the first part,the effect of matrine on the growth and proliferation of human hepatoma Hep G2 cells was studied.Human hepatoma Hep G2 cells were treated with matrine at different concentrations(0.0 mg/ml,0.2 mg/ml,0.4 mg/ml,0.8 mg/ml,1.6mg/ml),and cultured at different times(24H,48 H,After 72H),the cells were collected,and the proliferation inhibition effect of matrine on human liver cancer Hep G2 cells was examined by CCK8 proliferation assay.In the second part,we studied the radiosensitizing effect of matrine on human hepatoma Hep G2 cells.1.By combining the application of a certain concentration of matrine and radiotherapy on human hepatoma Hep G2 cells,the apoptosis and cell cycle distribution of each group were detected.Specifically,human hepatoma Hep G2 cells were divided into 4 groups,which were blank control group.Matrine group,irradiation group and irradiation + matrine group were used to detect apoptosis and cell cycle distribution of hepatoma cells by Annexin V-FITC/PI double staining flow cytometry,and to judge the early and late stage of matrine in hepatoma cells.Apoptosis and cell cycle have no effect;2.By combining a certain concentration of matrine and radiotherapy on human hepatoma Hep G2 cells,continue to culture 48 H and 72 H,and detect the MUC-1 protein in cells before and after matrine treatment by Western blotting(WB method).The expression of MUC-1 m RNA in matrine was analyzed by real-time fluorescent quantitative PCR(RT-PCR).It was judged whether matrine had any effect on MUC-1 protein in hepatoma cells.Results The first part studied the effect of matrine on the growth and proliferation of human hepatoma Hep G2 cells,and the results showed,Matrine inhibited the growth and proliferation of human hepatocellular carcinoma Hep G2 cells in a time-dose dependent manner.Within the experimental concentration range,with the increase of drug concentration and the prolongation of drug action time,the proliferation activity of human hepatocellular carcinoma Hep G2 cells gradually decreased,that is,the inhibition rate of proliferation of hepatocellular carcinoma cells gradually increased.IC50 values(half inhibitory concentration)of 24 H,48H and 72 H treated with Matrine were 0.6,0.41 and 0.38 mg/ml,respectively.The second part is to study the radiosensitizing effect of matrine on human hepatoma Hep G2 cells.The experimental results show that:1.The results of cell cycle distribution were detected by Annexin V-FITC/PI double staining flow cytometry.The G2/M phase cells had the strongest radiosensitivity,and the S phase cells were the second.Therefore,the ratio of G2/M phase and S phase cells was mainly compared.The cells were treated with low-cytotoxic matrine(0.2 mg/ml)medium for treatment of human hepatoma Hep G2 cells for48 H.The cell cycle results of flow cytometry showed that matrine was found in the matrine group and the blank control group.The G2/M phase cells in the group were slightly higher than the G2/M phase cells in the control group,but the statistical results were not significantly different(P>0.05).The S phase cell difference between the two groups was P=0.478(P>0.05),and the difference was also No significantness.Compared with the control group and the blank control group,the G2/M phase of the irradiated group was slightly increased compared with the control group,P=0.026,the statistical results were different(P<0.05),and there was no significant difference in the S phase.Compared with the matrine+ matrine group and the blank control group,the S phase cells of the matrine + irradiation group increased slightly,but there was no statistical difference(P>0.05).Compared with the irradiation+ matrine group and the simple irradiation group,it was found that the G2/M phase cells in the irradiation + matrine group were slightly decreased,and the S phase was slightly increased,the difference was statistically significant(P < 0.05),so it was the least sensitive.Compared with G0/G1 phase,there was no significant difference in G0/G1 phase(P=0.344),indicating no significant change in cell cycle.There was no significant difference in the cell distribution rate between G2/M phase,S phase and G0/G1 phase in the contrast + matrine group and the matrine group(P>0.05).It indicated that matrine had no significant effect on the cycle distribution of liver cancer cells.2.Flow cytometry detection of apoptosis showed that the early and late apoptotic rate of the irradiated group and the blank control group increased,and the number of normal living cells decreased(P<0.005).),indicating that radiation can induce apoptosis of liver cancer cells;compared with matrine group and blank control group,simple irradiation group and irradiation +matrine group,after matrine(0.2 mg/ml)is applied to human liver cancer Hep G2 cells,The early apoptotic rate and the late apoptotic rate were significantly increased,and the number of normal living cells was further decreased.The difference was statistically significant(P<0.001),indicating that matrine can induce apoptosis of hepatocarcinoma cells;In the irradiation + matrine group,the apoptotic rate of the irradiation + matrine group was higher than that of the simple matrine group(P < 0.001),indicating that matrine can further induce tumor cell apoptosis by synergistic radiation.3.The expression of MUC-1 protein by Western Blot(protein immunoblotting)showed that the expression of MUC-1 protein in the irradiated group was higher than that in the blank control group(P<0.001).It indicated that radiation could cause the increase of MUC-1 protein in liver cancer cells.Compared with the expression of 48 H and 72 H in different culture time,the expression level of72 H was higher than that of 48 H.Compared with the matrine group and the blank control group,it was found that the expression of MUC-1 protein was decreased after matrine treatment on human hepatoma Hep G2 cells,indicating that matrine can decrease the expression of MUC-1 protein in hepatoma cells;The expression of MUC-1 protein in 72 H culture time was lower in the expression of72 H at different culture time,indicating that the ability of matrine to inhibit the expression of MUC-1 protein was time-dependent.The longer the time,the more the inhibition ability was.Strong,the results were statistically significant(P< 0.001);Compared with matrine and blank control group,irradiation and irradiation + matrine group,it was found that the amount of MUC-1 protein was lower after matrine treatment of human hepatoma Hep G2 cells;comparison with matrine group and irradiation+ Sophora flavescens In the alkali group,irradiation + matrine group,it was found that the amount of MUC-1 protein in the irradiation + matrine group was lower,indicating that matrine can inhibit the expression of MUC-1 protein in human hepatoma Hep G2 cells alone,while matrine Combined with radiation,it can further inhibit the expression of MUC-1 protein in human hepatoma Hep G2cells(P<0.001),indicating that matrine can synergistically inhibit the expression of MUC-1 protein in human hepatoma Hep G2 cells.4.The expression of MUC-1 m RNA was detected by RT-PCR.It was found that the expression of MUC-1 m RNA in the irradiated group was higher than that in the blank control group(P<0.001),indicating that radiation can cause liver cancer.The increase of MUC-1 m RNA in the cells was compared with the expression of 48 H and 72 H at different culture time.The expression of 72 H was higher than that of 48 H.Compared with the matrine group and the blank control group,it was found that the expression of MUC-1 m RNA was decreased after matrine treatment on human hepatoma Hep G2 cells,indicating that matrine can decrease the expression of MUC-1 m RNA in hepatoma cells;comparing 48 H and 72 H The expression of MUC-1 m RNA in 72 H culture time was lower in the expression of two different culture time,indicating that the ability of matrine to inhibit the expression of MUC-1 m RNA was time-dependent.The longer the time,the stronger the inhibition ability.Statistically significant(P <0.001);Compared with matrine and blank control group,irradiation and irradiation + matrine group,it was found that the amount of MUC-1 m RNA was lower after matrine treatment of human hepatoma Hep G2cells;compared with matrine group and irradiation + matrine group,In the irradiation + matrine group,it was found that the amount of MUC-1 m RNA in the irradiation + matrine group was lower,indicating that matrine can inhibit the expression of MUC-1 m RNA in human hepatoma Hep G2 cells alone,and when matrine is combined with radiation,It can further inhibit the expression of MUC-1 m RNA in human hepatoma Hep G2 cells(P<0.001),indicating that matrine can synergistically inhibit the expression of MUC-1 m RNA in human hepatoma Hep G2 cells.The trend of expressing MUC-1 m RNA in each group was similar to the trend of detecting MUC-1 protein expression by WB method,and the results were consistent with the results of WB assay for detecting MUC-1 protein.Conclusion1.Matrine can inhibit the proliferation of tumor cells and induce apoptosis of tumor cells;2.The effect of matrine combined with radiotherapy on proliferation inhibition and apoptosis of human hepatoma Hep G2 cells is significantly higher than that of matrine alone or radiotherapy alone;Alkali has radiosensitizing effect on human hepatoma Hep G2 cells,and its mechanism may be related to inhibiting Hep G2 tumor cell proliferation,inducing tumor cell apoptosis and reducing MUC-1 protein and MUC-1 m RNA expression in human hepatoma Hep G2 cells,and regulating cell cycle.Redistribution may not matter.These results indicate that matrine can be used as a sensitizing drug for liver cancer radiotherapy,and it provides an experimental basis and theoretical basis for the in-depth study of matrine.Innovation A new Chinese herbal extract was tried to explore whether it can be used as a radiosensitizer for tumors.At present,the sensitization effect of matrine on radiotherapy is more common in clinical reports.There is no experimental verification and matrine sensitization mechanism.This experiment explored the possible mechanism of matrine combined with radiotherapy to inhibit the proliferation of hepatoma cells: induce apoptosis and inhibit the expression of MUC-1,which has certain guiding significance for clinical treatment. |
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