| Object:To observe the influence of exogenous heat shock protein90 inhibitor 17-DMAG on the growth,metastasis of TA2 mouse mammary cancer by the transplanted tumor model of highly metastatic TA2 mouse mammary cancer,and identify the role of heat shock protein90 in the growth,metastasis of TA2 mouse mammary cancer.Then further detect the expression of MMP2,MMP7,MMP9,α-Tubulin,integrinα4,integrinαL,integrinβ2,which involves in metastasis of tumors, in tumor tissue.To investigate the related mechanisms of inhibition of 17-DMAG to the metastasis of TA2 mouse mammary cancer.Method:1.To select 40 adult female TA2 mice,the high metastasis mammary cancer cells of TA2 mouse are transplanted to the left groin of mouse,1×10~5/each mouse,and randomly divided into medication treated and control group,20 TA2 mice per group. And to observe the active state of mice,growth of tumor every day after seeding cancer cells.Each mouse is given 17-DMAG 1mg by intraperitoneal injection after touching tumors,twice per week.And to measure the size of tumor and drawing growth curve of tumor.2.10 TA2 mice each group are sacrificed on the 14th(giving medicine 4 times),21th(giving medicine 6 times) day after giving medicine.The tissues of mouse transplanted tumors,live and spleen are collected completely and weighed.Some fresh tissues without necrosis are extracted RNA by TRIzol,and run RNA quantity by ultraviolet spectrometry for Real-time PCR.Others with the liver,spleen of mouse are fixed by 10%formalin for H&E dyeing and immunohistochemistry staining.To detect the expressions of MMP2,MMP7,MMP9,α-Tubulin,integrinα4,integrinαL, integrinβ2 of tumor tissue in the groups at the protein and mRNA level by immunohistochemistry and Real-time PCR.Result:1.The growth information of tumor of two groups:The tumor was touched in the mouse groin on the 4th day after seeding cancer cells.The mice,especially mice of control group,gradually showed up bad active state and lower body surface temperature following the increase of number of medicine administration and extension of growth of tumor.Some mice were dead of fast growth speed of tumor,heavy metastasis or disruption of organs in the process of experiment. Finally the number of collected sample is 29(16 of medicine treated group,13of control group).2.The growth speed of tumor of medicine treated group are obviously lower than that of control group after 17-DMAG medicine administration,especially in the early stage.The average volume of tumor of medicine treated group is lower than that of control group on the 14th day after medicine administration,the difference has statistical significance(P<0.05),while the difference has no statistical significance on the 21th day after medicine administration.3.The metastasis rate of live,spleen of mice respectively is 31.25%,81.25%in medicine treated group,while both are 92.31%in control group.The metastasis rate of liver of mice of medicine treated group is obviously lower than that of control group,the difference has statistical significance(P<0.05),while the difference of metastasis rate of spleen has no statistical significance.Though the rate of spleen metastasis of medicine treated group is not lower,the structure of spleen is approximately normal,the area of metastasis is obviously small,and sporadic single tumor giant cell in the lymphocyte under light microscope.4.The difference of weight of tumor and spleen between two groups have statistical significance(P<0.05),while the difference of weight of liver has no statistical significance on the 14th day after medicine administration.The difference of weight of spleen has statistical significance(P<0.05) between the two groups on the 21th day after medicine administration.5.The expression of MMP2,MMP7,MMP9,α-Tubulin,integrinαL,integrinβ2 of tumor of medicine treated group(respective expression: 0.2133±0.1181,0.0305±0.0273,0.0833±0.0606,0.145±0.055,0.199±0.033,0.272±0 .042) is lower than that of control group(respective expression:0.4621±0.2338, 0.2193±0.1430,0.2300±0.0510,0.450±0.109,0.307±0.027,0.389±0.099),the difference all have statistical significance(P<0.05).while the expression of integrinα4 of medicine treated group(0.13±0.052) is higher than that of control group(0.12±0.040),the difference has no statistical significance.6.The expression of MMP2,MMP9,integrinαL,integrinβ2 mRNA of tumor of medicine treated group(0.0013±0.0004,1.034±0.784,0.0020±0.0011, 0.0094±0.0037) is lower than that of control group(0.0010±0.0003,2.318±1.294, 0.0037±0.0023,0.0183±0.0072),the difference all have statistical significance (P<0.05),while the difference of expression of MMP7,integrinα4 mRNA between two groups have no statistical significance.Conclusions:1.17-DMAG can obviously inhibit the growth and metastasis of mice mammary cancer of TA2 in the early stage.2.The results of the 17-DMAG interfered experiment suggest that the binding of 17-DMAG and HSP90 can actively suppress the biological function of HSP90, and degrade the metastasis rate of liver and spleen of TA2 mouse mammary cancer,we presume that HSP90 can promote the growth and metastasis of mammary cancer of TA2 mouse.3.The expression of MMP2,MMP7,MMP9,α-Tubulin,integrinαL,integrinβ2 are down regulation in the 17-DMAG treated group,we presume that the mechanisms of inhibition of 17-DMAG to metastasis of TA2 mouse mammary cancer could be its influence on the expression of MMP2,MMP7,MMP9,α-Tubulin,integrinαL, integrinβ2 by binding to HSP90. |
PDF Full Text Request