Studies On The Vascular Effects And EMT Induction Of Arsenite

Chronic endemic arsenism via drinking water, is a chronicity cumulative toxicosis disease and becomes an international health hazard. In order to reduce the adverse health effects of arsenism, the central and local governments of China have provided significant funds to change water levels of As and at the same time take general measures to "reduce arsenic intake, remove arsenic from the body and treat the patients". However, the pathogenesis of arsenism is not clear and carcinogenesis of arsenism is very complicated.we designed a series of experiments to detect the effect of sodium arsenite on the Angiogenesis and carcinogenesis and the possibly involved mechanisms.Part I in Vivo and in Vitro Studies on the Effect of Sodium Arsenite on the AngiogenesisObjective:The studies were carried out to explore the effect of sodium arsenite on the angiogenesis to provide theoretical explanation and practical measurement for arsenism. Methods:The angiogenesis of Chorioallantoic membrane (CAM) were used as a model to evaluate in vivo effects of arsenic on it. The proliferation of human umbilical vein endothelial cells (HUV-EC) was used as an in vitro model to evaluate effects of arsenic on human vascular cells. Cell proliferation was assayed by CCK-8 kit. The mRNAs were analyzed by real time quantitative RT-PCR. Results:Arsenic showed a bilateral effect on the angiogenesis in CAM model:the stimulating effect when exposed to low-level and suppressing effects when exposed to high-level arsenic. Sodium arsenite inhibited proliferation of HUV-EC in a dose-dependent manner when exposed to it in levels applied. Meanwhile, the mRNAs of c-Jun and c-Myc were decreased following exposure to sodium arsenite. Conclusion:Sodium arsenite inhibits proliferation of HUV-EC through the down regulation of c-Jun and c-Myc. suggesting they are the major signals for arsenic to exert its suppression on the proliferation of HUV-EC.Part II Effects of Sodium Arsenite on the NMuMGObjective:To study the effects of Sodium Arsenite on NMuMG. Methods:Utilize microscopy observe cell morphology;CCK-8 kit detect cell growth:Annexin V/PI with flow cytometry detect cell apoptosis;Utilized RT-PCR detecting mRNA level of relative genes. Results:compared with un-treated group.treated group cells' appearance was changed and was irregular, cell proliferation activity was decreased significantly; apoptotic cells increased; RT-PCR showed that the level of c-myc was decreased,and the expression of P21 was increased. Conlusion:NaAsO2 can change the appearance of NMuMG,inhibite the cell proliferation, promote apoptosis by reducing the expression of c-Myc and increasing the expression of P21.PartⅢConstruction of model of TGF-βInduced Transdifferentiation of Mammary Epithelial Cells to MesenchymalObjective:To construct the model of EMT and provid theory foundation for arsenic carcinogenesis. Methods:NMuMGs were cultured.when it growed up to 50%,3% serous medium were changed,and different concentration of TGF-βwere added into medium after 24 hour.when TGF-(3 working on the NMuMGs for 24 hours,observing the cell morphology, detecting the E-cadherin and Vemintin through immunocytochemical staining and mRNA expression through RT-PCR.Results:un-induced group cells showed cubical epithelial cell and induced group cells showed spindal mesenchymal cell,the immunocytochemical staining showed thatun-induced group E-ca is positive,Vimentin is negative;induced group E-ca is negative, Vimentin is positive strongly. Meanwhile, the mRNAs of E-ca was increased and Vimentin was decreased following exposure to 5ng/ml TGF-β1.Conclusion:Succed to construct the model of EMT.PartⅣNaAsO2 Induced Transdifferentiation of Mammary Epithelial Cells to MesenchymalObjective:The studies were carried out to explore the mechanism of arsenism carcinogenesis. Methods:NMuMGs were cultured.when it growed up to 50%,3% serous medium were changed.and 5ng/ml TGF-(3 and 10 u M NaAsO2 were added into medium after 24 hour.when these medium working on the NMuMGs for 24 hours, utilizing microscopy observe cell morphology,the immunocytochemical staining detect the E-cadherin and Vemintin.RT-PCR detect mRNA expression. Results:as the 5ng/ml TGF-βgroup,the cells of 10μM NaAsO2 group showed from cubical epithelial cell to spindal mesenchymal cell,through the immunocytochemical staining,10μM NaAsO2 group E-cadherin is negative, Vimentin is positive;Meanwhile, the mRNAs of E-cadherin was decreased and Vimentin was increased following exposure to 10μM NaAsO2 So NaAsO2 succeded to induce NMuMGs EMT.To detect the molecular mechanism,RT-PCR results showed that the mRNA expression of HMGA2 and zinc-finger protein slug were elevated.Conclusion:Sodium arsenite can induce NMuMGs EMT which maybe the mechanism of arsenism carcinogenesis.And they work through HMGA2 pathway.