| Objective:To establish a subcutaneous transplanted tumor model of human lung cancer in nude mice,to observe the effects of ketogenic diet and combined radiotherapy and chemotherapy on the growth of subcutaneous transplanted tumor in nude mice and to study its mechanism.Methods:A549 cells in logarithmic phase were digested with 0.25%trypsin,harvested and counted,the concentration was adjusted to 1.0×10~7ml,and the cell suspension with the adjusted concentration was injected into the right lower limb skin of nude mice with 1ml syringe,each lnoculate0.2ml,inoculate a total of 20 animals.The established nude mouse lung cancer transplanted model was randomly divided into 4 groups,5 in each group:standard diet group(SD group),ketogenic diet group(KD group),standard diet+cisplatin+radiotherapy group(SD+DDP+RT group)and ketogenic diet+cisplatin+radiotherapy group(KD+DDP+RT group).Nude mice in SD group and SD+DDP+RT group were fed with standard diet,while nude mice in KD group and KD+DDP+RT group were fed with ketogenic diet.Observe and record the diet,mental state,stool and activity of nude mice daily.On the day of grouping,body weight,blood glucose,blood ketone,and long and short diameters of transplanted tumors were measured.After every 5 days.Observe the weight change of nude mice during the experiment,calculate the approximate volume of the transplanted tumor,and draw the tumor growth curve.When the tumor volume of nude mice in SD+DDP+RT group and KD+DDP+RT group reaches to 100?200mm~3,the nude mice were treated with radiotherapy with a total dose of 4 Gy,and cisplatin was injected every 3days for 4 times.On the 30th day after inoculation,the eyeballs were removed and the blood was taken and sent to the laboratory for four tests of blood lipids.After the nude mice were killed,the transplanted tumor tissue was taken out for HE staining,and the morphological changes of tumor cells were observed.Western blot and immunohistochemical methods were used to detect the protein expression of 4-HNE in the transplanted tumor of nude mice.Results:1. From 8 to 10 days after cell inoculation,the solid tumor could be seen with the naked eye,which was oval.All the 20 nude mice formed tumors,and the tumor formation rate was 100%.During the whole experiment,there was no obvious abnormality in diet and mental state of nude mice in each group.2. On the 10th day of cell inoculation,there was no significant difference in body weight between the SD group,KD group,SD+DDP+RT group,KD+DDP+RT group(P>0.05).Since the 20th day of inoculation,there was significant difference in body weight between the four groups of nude mice(P<0.05).The body weight of nude mice in KD group and KD+DDP+RT group was lower than SD group and SD+DDP+RT group,and the differences were statistically significant(P<0.05).There was no significant difference in pairwise comparison between the other groups(P>0.05).3. There was no significant difference in the tumor volume of the four groups of nude mice at the 10th day and the 15th day after cell inoculation(P>0.05).On the 20th day,25th day,and 30th day after inoculation,there were significant differences in the volume of transplanted tumor among the four groups(P<0.05).Compared with the SD group,the tumor volume of the KD group,SD+DDP+RT group,and KD+DDP+RT group decreased,and the differences were statistically significant(P<0.05).Compared with the SD+DDP+RT group,the tumor volume of the KD+DDP+RT group was reduced,and the difference was statistically significant(P<0.05).On the 25th and 30th days of inoculation,the volume of the transplanted tumors in the nude mice of the KD+DDP+RT group was smaller than that of the KD group,with a significant difference(P<0.05).There was no significant difference in pairwise comparison between the other groups(P>0.05).4. On the 10th day of cell inoculation,there was no significant difference in blood glucose between the SD group,KD group,SD+DDP+RT group and KD+DDP+RT group(P>0.05).On the 15th day,20th day,and 30th day after inoculation,the blood glucose levels of the four groups of nude mice were significantly different compared with each other(P<0.05).The blood glucose levels of nude mice in KD group and KD+DDP+RT group were lower than those in SD group and SD+DDP+RT group,and the differences were statistically significant(P<0.05).There was no significant difference in pairwise comparison between the other groups(P>0.05).On the 25th day of inoculation,there was a statistically significant difference in blood glucose between the four groups of nude mice(P<0.05).Except that the difference between KD group and SD+DDP+RT group was not statistically significant,the differences between other groups were statistically significant(P<0.05).5. On the 10th day of cell inoculation,there was no significant difference in blood ketone values between the SD group,KD group,SD+DDP+RT group,and KD+DDP+RT group(P>0.05).On the 15th day,20th day,25th day,and30th day of inoculation,the differences in blood ketone values of the four groups of nude mice were statistically significant(P<0.05).KD group and KD+DDP+RT group had higher blood ketone values in nude mice than SD group and SD+DDP+RT group,the differences were statistically significant(P<0.05).On the 25th and 30th days of inoculation,the difference between the KD+DDP+RT group and the KD group was statistically significant(P<0.05).There was no significant difference in pairwise comparison between the other groups(P>0.05).6. On the 30th day after the cells were inoculated,the eyeballs were taken for blood to measure the levels of CHOL,HDL,TG and LDL in nude mice of different treatment groups.There was a statistically significant difference between the measurements of the four groups of nude mice(P<0.05).KD,KD+DDP+RT groups of nude mice had higher levels of CHOL,HDL,and LDL than SD and SD+DDP+RT groups,TG levels were lower than those of SD and SD+DDP+RT groups.The differences were statistically significant(P<0.05).There was no significant difference in pairwise comparison between the other groups(P>0.05).7. Western Blot results showed that the relative expression levels of 4-HNE protein in SD group,KD group,SD+DDP+RT group,and KD+DDP+RT group were 1.00±0.03,1.45±0.10,1.52±0.07,and 2.49±0.06.The difference between the four groups was statistically significant(P<0.05).Compared with SD group,KD group,SD+DDP+RT group,KD+DDP+RT group protein expression increased,the differences were statistically significant(P<0.05).Compared with KD group and SD+DDP+RT group,KD+DDP+RT group protein expression increased,and the differences were statistically significant(P<0.05).There was no significant difference in pairwise comparison between the other groups(P>0.05).8. The results of immunohistochemical methods showed that the median 4-HNE immune response scores of SD group,KD group,SD+DDP+RT group,and KD+DDP+RT group were 2,4,4,6.The difference between the four groups was statistically significant(P<0.05).Compared with SD group,KD group,SD+DDP+RT group,KD+DDP+RT group protein expression increased,the differences were statistically significant(P<0.05).Compared with KD group and SD+DDP+RT group,KD+DDP+RT group protein expression increased,and the differences were statistically significant(P<0.05).There was no significant difference in pairwise comparison between the other groups(P>0.05).Conclusions:1.Ketogenic diet can inhibit the growth of transplanted tumor in nude mice,and the inhibitory effect of ketogenic diet combined with radiotherapy and chemotherapy is more obvious.The anticancer mechanism is related to the increased expression of 4-HNE in tumor cells.2.Ketogenic diet and radiotherapy and chemotherapy have a synergistic effect on enhancing the oxidative stress of tumor cells,which further improves the effect of radiotherapy and chemotherapy in the heterogeneous model of lung cancer. |
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