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目的研究在建立抗砷细胞模型CAsE-人骨髓间充质干细胞(hBMSCs)过程中,低剂量砷长期暴露对hBMSCs分化的作用。方法常规条件培养hBMSCs,实验组用1μmol/L的亚砷酸钠连续诱导hBMSCs≥12周,采用48h急性砷中毒实验检测细胞抗砷性的产生;细胞集落形成实验分析CAsE-hBMSCs的增殖能力;应用RT-PCR、免疫细胞化学技术检测砷对Oct-4表达的影响;RT-PCR检测砷对ABCG2表达的影响。结果用1μmol/L的亚砷酸钠连续诱导12周后,hBMSCs获得抗砷性,砷诱导hBMSCs的LC50为35.59μmol/L,平行培养的hBMSCs的LC50为18.04μmol/L;与对照组比较,CAsE-hBMSCs不具有恶性增殖能力;Oct-4基因在第4代、第18代及砷诱导4周的hBMSCs中均有表达,但在亚砷酸钠诱导12周、15周后未检测到Oct-4的表达;Oct-4蛋白在第4代hBMSCs表达阳性,砷诱导15周的CAsE-hBMSCs中呈弱阳性表达,阳性颗粒分布在胞质内;实时定量结果显示ABCG2在砷诱导15周的hBMSCs中的表达明显低于对照组(P<0.001)。结论长期低剂量NaAsO2可诱导人骨髓间充质干细胞分化。
Objective To study the effect of low dose arsenic exposure on the differentiation of hBMSCs during the establishment of anti-arsenite cell line CAsE-human bone marrow mesenchymal stem cells (hBMSCs). Methods hBMSCs were cultured in routine conditions. The hBMSCs were continuously induced by 1μmol / L sodium arsenite for 12 weeks in the experimental group, and the arsenism resistance was detected by 48h acute arsenism. The proliferation of CAsE-hBMSCs was analyzed by colony formation assay. The effect of arsenic on the expression of Oct-4 was detected by RT-PCR and immunocytochemistry. The effect of arsenic on the expression of ABCG2 was detected by RT-PCR. Results After 12 weeks of continuous induction with 1 μmol / L sodium arsenite, the hBMSCs obtained arsenic resistance. The LC50 of arsenic-induced hBMSCs was 35.59 μmol / L and the LC50 of parallel cultured hBMSCs was 18.04 μmol / L. Compared with the control group, CAsE-hBMSCs had no malignant proliferative ability. Oct-4 gene was expressed in the 4th, 18th generation and arsenic-induced hBMSCs for 4 weeks. However, OctA-4 gene was not detected after 12 weeks and 15 weeks induction of sodium arsenite -4 expression; Oct-4 protein in the fourth generation of hBMSCs positive expression, arsenic-induced 15 weeks of CAsE-hBMSCs were weakly positive expression of positive particles distributed in the cytoplasm; real-time quantitative results showed ABCG2 in the arsenic-induced 15-week hBMSCs expression was significantly lower than the control group (P <0.001). Conclusion Long-term, low-dose NaAsO2 induces human bone marrow mesenchymal stem cell differentiation.