Experimental Study Of ADAM17-shRNA Transfected Bmscs In The Treatment Of Breast Cancer Of Nude Mice

Objectives Use breast cancer bearing tumor model in nude mice to investigate the antitumor effect and mechanism of mediated bone marrow derived mesenchymal stem cells(BMSCs)with ADAM17-sh RNA transfected by lentivirus mediated.Methods The BMSCs of male SD rats aged 4w were isolated,cultured and amplified by adherence screening method,and the BMSCs with high purity,strong proliferation ability and sufficient quantity could be obtained when spread to the third generation.ADAM17-sh RNA carrier was designed and compounded according to the specificity of ADAM17 gene,using lentivirus mediated to transfect the ADAM17-sh RNA to the third generation of BMSCs.The transfection rate of lentivirus mediated ADAM17-sh RNA in BMSCs was observed by laser scanning confocal microscopy.Nude mice were inoculated subcutaneously with MCF-7 cells,and 0.2 ml for each mouse(5x107/ml)were implanted in the right side of nude mice.The experiment were randomly divided into 5 groups: control group(blank PBS),low dose BMSCs group(1x106/ml BMSCs),high dose BMSCs group(5x106/ml BMSCs),low dose transfection group(1x106/ml BMSCs transfected ADAM17-sh RNA)and high dose transfection group(5x106/ml BMSCs transfected ADAM17-sh RNA).BMSCs transfected ADAM17-sh RNA was injected by tail vein,0.1ml for each time,and repeated every 3 days,and a total of 5 times.The general situation and the growth of tumors were observed in nude mice bearing tumor in each group,and at the end of the experiment the tumor volume and the tumor inhibition rate were calculated,morphology of tumor were observed by HE staining,and the expression ADAM17,EGFR and Ki-67 of tumor was detected by immunohistochemistry.Results 1 The observation of isolation,cultivation and amplification of the third generation of BMSCs cells under inverted phase contrast microscope showed that the morphology of the cells was consistent,and the spindle shape and the horn shape were the majority.The cells showed a swirl like disaster colony distribution with less impurity cells,and can obtain BMSCs with high purity,strong proliferation ability,and a great quantity.2 Laser scanning confocal microscopy showed that lentivirus mediated ADAM17-sh RNA could be successfully transferred into BMSCs,and the transfection rate was over 85%.3.At the seventh day after nude mice were inoculated subcutaneously with MCF-7 cells,the tumor rose on the surface of the skin,which means the nude mice model of breast cancer was successfully constructed.4.After the transfected ADAM17-sh RNA were injected into nude mice by the tail vein,it was observed that the high dose transfection group and the low dose transfection group had good spirit and the general condition was good.Nude mice of high dose BMSCs group had general spirit,decreased food intake and no loss of weight,while nude mice of the control group and the low dose BMSCs group had poor mental health,poor general condition,weight loss,and cachexia.After 16 days of treatment,Nude mice skin cancer surface was appear a small number of necrosis,ulceration,Showing a number of nodular,touch hard,The general observation,The tumor size of the high dose transfection group,the low dose transfection group and the high dose BMSCs group was significantly lower than the control group,However,the small dose of BMSCs group and the control group tumor volume is basically no difference.5 By measuring the size of tumor volume changes of nude mice,with the prolonging of feeding time,the tumor volume of high dose transfection group,low dose transfection group and high dose BMSCs group increased slowly,The anti-tumor effect is remarkable,while the tumor volume of the control group and low dose BMSCs group increased fast;At the end of the experiment(30d),The tumor volume of each group was compared,Large dose transfection group,low dose transfection group and high dose BMSCs group were smaller than the control group,The tumor volume of the nude mice in large dose transfection group,low dose transfection group and high dose BMSCs group was smaller than the control group,The difference was statistically significant(P<0.05),While the volume of tumor in nude mice was not changed between the control group and the low dose BMSCs group,The difference was statistically significant(P>0.05);Compared with the control group,the maximum inhibition rate of high dose transfection group,low dose transfection group,high dose BMSCs group and low dose BMSCs group was 66.33%,52.13%,37.4% and 2.31% respectively.6 HE staining showed that the tumor tissue showed typical human breast infiltrating ductal carcinoma,and carcinoma cells showed typical glands like arrangement,interstitial infiltration,and the formation of a large number of typical breast cancer nests.The large area of necrosis of tumor tissue was seen in the high dose transfection group,and the necrotic area cells were disintegrated and the cell structure disappeared.7 Immunohistochemical staining showed,The expression of ADAM17,EGFR and Ki-67 protein in 5 groups were compared,Differences were statistically significant(F=93.970,P=0.000;F=84.684,P=0.000;F=58.024,P=0.000),Compared with the control group,the expression of ADAM17,EGFR and Ki-67 protein in the high dose transfection group,the low dose transfection group and the high dose BMSCs group decreased(P<0.05),And the expression of three protein in the high dose transfection group and the low dose transfection group were the most significant;There was no significant change in the expression of ADAM17,EGFR and Ki-67 between the control group and the low dose BMSCs group(P=0.324>0.05;P=0.240>0.05;P=0.484>0.05).Conclusion BMSCs transfected by lentivirus mediated ADAM17-sh RNA can inhibit the expression of ADAM17,EGFR and Ki-67 of nude mice with breast cancer,and played an anti-tumor effect of breast cancer in nude mice.