The Detection Of Arsenic Resistance Relative Genes Using Human Bone Marrow Mesenchymal Stem Cells

Objective: To set up the arsenic resistance cell model by chronic arsenite sodium exposure of human Bone marrow Mesenchymal Stem Cells and detect the arsenic resistance relative genes .Methods: Human Bone marrow Mesenchymal Stem Cells (hBMSCs) was isolated and cultured under commom conditions. HBMSCs was exposed at sodium arsenite(AsIII) for over 14 weeks and the control cells was parallelly cultured with same conditions. 48 hours acute arsenite toxicity test was performed to assay cell living rate. Then, the phenotype of hBMSCs was detected by Immunofluorescence Staining and Fluorescence- activated cell sorting before and after Arsenite induction. We used Flow Cytometry to assay the cell cycle and apoptosis. Furthermore, Real time-PCR was performed to identify the expression of metallothionein gene MT1E,multidrug resistance protein gene MRP1 and human arsenic resistance gene HARRG. Finally, the cell malignant transformation was detected by soft-agar experiment. When the arsenic resistance was obtained, total RNA was extracted and purified to hybridize with Affymetrix UC133 plus 2.0 gene microarray and the function of distinctive genes was analyzed by bioinformatics.Results: Acute low level arsenite exposure promotes cell proliferation but chronic not. HBMSCs primarily exhibited arsenic resistance when exposed under 1μmol/L arsenite sodium over 10 weeks, and this consequence was obvious when exposed over 14 weeks. The phenotype of hBMSCs was the same as the control after 14 weeks arsenite exposure but the cell cycle changed dramatically. Over 5μmol/L acute arsenite exposure can induce cell apoptosis. MT1E, MRP1 and HARRG was up- regulated in arsenite exposed cells during some time. HBMSCs were not malignant transformative because the hBMSCs couldn't grow in the two layer soft-agar after chronic arsenite exposure. Finally, When hybridized with Affymetrix UC133 plus 2.0 gene microarray, we have detected 1876 distinctive genes ,while 1120 genes were up-regulated and 756 genes were down-regulated.Conclusion: Long and low level arsenite exposure induced arsenic resistance in human Bone marrow Mesenchymal Stem Cells and we have detected 1876 arsenic resistance related genes.