慢性砷暴露人骨髓间充质干细胞的生物学特性观察

目的 观察抗砷细胞模型慢性砷暴露人骨髓间充质干细胞(CAsE-hFBMSCs)的生物学特性,探讨长期低剂量砷暴露对人骨髓间充质干细胞(hFBMSCs)的影响.方法 常规条件培养hFBMSCs,48 h急性砷毒性实验MTS法检测不同剂量砷刺激条件下0(对照)、0.25、0.50、1.00、2.00、4.00、8.00、20.00、40.00、80.00、120.00μmol/L]第2代(P2)hFBMSCs细胞生存率,根据检测结果用1.00μmol/L亚砷酸钠刺激hFBMSCs 14周,建立抗砷细胞模型CAsE-hFBMSCs,作为实验组,同时设立对照组,并采用荧光激活细胞分选术鉴定诱导前后细胞表型,流式细胞术检测第2、9、16代(P2、P9、P16)细胞周期,软琼脂克隆形成实验检测细胞是否发生恶性转化.结果 <10μmol/L时,急性砷刺激促进hFBMSCs增殖,而≥10μmol/L时,急性砷刺激则抑制细胞的生长.14周时实验组半数致死剂量(LC_(50))为(89.42±0.64)μmol/L,对照组为(52.48±0.71)μmol/L,组间比较差异有统计学意义(t=123.89,P<0.05);抗砷细胞模型CAsE-hFBMSCs的细胞表型CD34、CD45表达阴性,CD29、CD90、CD166表达阳性,与对照组比较细胞表型未见明显改变;CAsE-hFBMSCs的细胞周期变化较大,与对照组(8.44±0.45)%、(9.14±0.14)%、(82.42±0.60)%]比较,P2实验组G2/M期(17.72±5.47)%]和S期细胞(25.34±3.36)%]增加,G0/G1期细胞减少(56.96±8.83)%],P16细胞周期恢复至与对照组相近的水平:软琼脂克隆形成实验中,抗砷细胞CAsE-hFBMSCs未见克隆形成.结论 长期低剂量砷刺激对hFBMSCs生物学特性无明显影响.

Biological characteristics of arsenic resistance cell chronic arsenic exposure human bone marrow mesenchymal stem cells

Objective To study the biological characteristics of arsenic resistance cell model chronic arsenic exposure human bone marrow mesenchymal stem cells (CAsE-hFBMSCs) and discuss the consequence of chronic arsenite exposure to human mesenchymal stem cells (hFBMSCs). Methods hFBMSCs cultivated under general conditions,hFBMSC cell survival rate was detected in 48 hours with arsenite toxicity test under different doses arsenic 0(control),0.25,0.50,1.00,2.00,4.00,8.00,20.00,40.00,80.00,120.00 μmol/L]of the fist 2-generation(P2). According to the test results,1.00 μmol/L sodium arsenite was chosen to stimulate hFBMSCs for 14 weeks as experimental group,simultaneous 0 μmol/L sodium arsenite as the control group. And then,the phenotype was detected by fluorescence-activated cell sorting,and the cell cycle by flow cytometry. Finally,the cell malignant transformation was detected by soft-agar assay. Results Arsenite low than 10 μmol/L promoted cell proliferation,but inhibited cell proliferation when exceeding 10 μmol/L. Half of the lethal dose (LC_(50)) in experimental and control groups were (89.42±0.64),(52.48±0.71)μmol/L. The difference between two groups was statistically significant(t = 123.89,P < 0.05). The phenotype of CAsE-hFBMSCs was CD29,CD90,CD166 positive and CD34,CD45 negative. The phenotype of CAsE-hFBMSCs was the same as the control. Comparing to control group(8.44±0.45)%,(9.14μ0.14)%,(82.42±0.60)%],G2/M phage(17.72±5.47)%]and S phage (25.34±3.36)%]cell increased,G0/G1 phage(56.96±8.83)%]cell decreased in P2 CAsE-hFBMSCs. The cell cycle became nearly the same as the control group after adaption. CAsE-hFBMSCs did not show clone formation in soft agar clone formation assay. Conclusion Long last and low level exposure to arsenite does not influence the biologic features of hFBMSCs.