| BackgroundGastric cancer is one of the cancers with the highest morbidity and mortality in China.It is easily confused with other chronic diseases in the early stage.Therefore,when diagnosed,most of them are advanced patients,and there are no completely radical medical means at present.With the advent of cell immunotherapy drugs approved by the Food and Drug Administration(FDA),cell immunotherapy has become a hobby of cancer researchers and doctors.At present,chimeric antigen receptor T cells(CAR-T)have attracted much attention,and different targets and receptors have been studied and validated one after another.However,the study of chimeric antigen receptor NK cells(CAR-NK)is relatively rare,and its effect on solid tumors of gastric cancer has been rarely reported.In addition,Mucin 1(MUC1)is a large molecular weight glycoprotein,which exists in normal glandular epithelium and is over-expressed on the surface of various tumors and in gastric cancer tissues.Therefore,this study mainly focused on the inhibitory effect of NK cells modified with chimeric antigen receptor targeting MUC1 on gastric cancer.ObjectiveTo investigate the inhibitory effect of targeting MUC1 CAR-NK cells on xenograft tumor model of gastric cancer in mice.MethodsHuman gastric cancer cells(MGC-803 cells)with volume of 0.2 ml and concentration of 1~*10~5/ml were injected into the axilla of the right limb of BALB/c(nu)nude mice to construct a gastric cancer-nude mice model.The mice were randomly divided into six groups:control group,experimental group 1,experimental group 2,experimental group 3,experimental group 4 and experimental group 5.The mice were observed every day after inoculation.When the tumor volume of mice grew to about 100 mm~3(about 8th day after inoculation of MGC-803 cells),the length and diameter of the right limb nodules were measured with vernier caliper,and the weight of each mouse was weighed.The control group every 3 days was then given normal NK cells with a volume of 0.2 ml and a concentration of 1~*10~5/ml by tail vein.The experimental group 1-5 every 3 days was given targeting MUC1 CAR-NK cells immunotherapy by tail vein,respectively.The corresponding cell concentrations were 1~*10~5,1~*10~6,1~*10~7,1~*10~8 and 1~*10~9 MUC1CAR-NK/ml,and the injection volume was 0.2 ml.The length and diameter of the tumors and the weight of the mice were recorded every three days,and their diet and mental state were observed every day.From the day of immunotherapy with MUC1 CAR-NK cells,the mice were sacrificed on the 18th day after orbital blood collection.The weight of the mice after exfoliation and the weight of the tumors were weighed at the same time.Finally,the inhibition rate and the growth rate of the mice were calculated.ResultFirst,from the behavioral characteristics,the mice in the experimental groups treated with MUC1 CAR-NK cells were more active in mental state than those in the control group,and the decrease of appetite was not obvious.Second,from the visual treatment effect,compared with the control group,the tumor weight and volume of each experimental group were significantly reduced,with statistical difference(P<0.01);the inhibition rate of each experimental group was significantly increased,the inhibition rate was 51.03%,52.47%,46.72%,46.75%and 45.51%,respectively;the growth rate of mice in each experimental group was significantly increased,with a unified increase.The growth rate of mice in group 1,2 and 3 was significantly higher than that in group 4 and 5(P<0.05).At different time points,the tumor volume of mice showed that the tumor volume of each group increased gradually with the prolongation of the growth time of mice.Among them,the tumor volume of the control group increased continuously with the growth time,and the growth rate was faster;while the experimental groups showed a relatively flat growth with the growth time of the mice.In addition,after the treatment of MUC1 CAR-NK cells on the 8th day,there were significant differences between the experimental groups and the control group at the same time point of administration(P<0.01).At different time points,the weight of mice showed that:after the 8th day of administration,the weight of mice in the 1st,2nd and 3rd groups increased rapidly in a short time,then increased slowly;the weight of mice in the 4th and 5th groups kept a slow growth,and slightly decreased after 23rd day;while the weight of mice in the control group showed a continuous decline trend.And the rate of decline increases with time.Third,the results of inflammatory indexes showed that the levels of IL-6,IL-10 and TNF-alpha in serum of mice in each experimental group were significantly reduced,compared with the control group(P<0.05),and the above indexes in the experimental groups 1,2 and 3 showed a downward trend compared with the experimental groups 4 and 5,but there was no statistical difference(P>0.05).Fourth,the results of HE staining showed that MUC1 CAR-NK cells had a significant killing effect on tumor cells compared with the control group,while Tunel staining showed that compared with the control group,the number of apoptotic cells in each experimental group increased significantly(P<0.05);and compared with the experimental group 1,2and 3,the number of apoptotic cells in Experiment 4 and 5 groups withered significantly(P<0.05).The number of deaths decreased,but there was no statistical difference(P>0.05).Fivth,immunohistochemical results showed that the expression of Caspase-3 and Bax protein increased significantly,while the expression of Bcl-2 protein decreased significantly(P<0.05),and the expression of Caspase-3 and Bax protein decreased in groups 1,2 and 3 compared with groups 4 and 5.The expression of Bcl-2 protein showed a certain upward trend,but there was no statistical difference(P>0.05).ConclusionFisrt,targeting MUC1 CAR-NK cell has a significant inhibitory effect on the solid tumor growth of MGC-803 cells,with a wide concentration range and a high safety factor.Second,the optimal treatment concentration is 1~*10~6 MUC1 CAR-NK/ml.It provides experimental basis for the follow-up study of immunotherapy of gastric cancer. |
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