三氧化二砷调控再生障碍性贫血患者BM-MSC成脂/成骨分化异常的实验研究

Regulatory Effect of As_2O_3 on Imbalance between Adipogenic and Osteogenic Differentiation of BM-MSC from Patients with Aplastic Anemia

本研究旨在探讨三氧化二砷(As2O3)对再生障碍性贫血(aplastic anemia,AA)患者骨髓间充质干细胞(BM-MSC)成脂与成骨分化失衡的调控作用。分离纯化AA患者BM-MSC,分别置于成脂与成骨诱导分化培养体系,在相应培养体系中分别加入As2O3、环孢霉素A(Cs A)、As2O3联合Cs A及不加药,As2O3的药物浓度为0.001μmol/ml,Cs A的药物浓度为2.5 mmol/ml。将细胞相应分为As2O3组、Cs A组、联合组和对照组,采用细胞形态学观察、油红O染色、Von-Kossa染色及RT-PCR等检测方法,检测相应分化标志。结果显示,在向成脂诱导分化方面,As2O3组的脂肪细胞分化率为(18.3±1.9)%,低于Cs A组的(91.8±2.7)%及对照组的(92.1±1.2)%(P<0.05),而与联合组的(8.3±1.9)%相比,则未见明显差异(P>0.05),但Cs A组的脂肪细胞分化率要高于联合组(P<0.05),Cs A组与对照组相比则未见明显差异。RT-PCR检测结果显示,As2O3组的LPL-mRNA表达水平分要低于Cs A组及对照组(P<0.05),而与联合组相比,则未见明显差异(P>0.05),但Cs A组的LPL-mRNA表达水平分要高于联合组(P<0.05)。通过Von-Kossa染色检测发现,在成骨诱导培养14 d后,在As2O3组、联合组可见明显钙盐沉积,而Cs A组及对照组则未观察到明显钙盐沉积现象。RT-PCR检测结果显示,As2O3组的OST-mRNA表达水平要高于Cs A组及对照组(P<0.05),而与联合组相比,则未见明显差异(P>0.05),但Cs A组的表达水平要低于联合组(P<0.05)。结论:As2O3可以抑制AA患者BM-MSC向脂肪细胞分化,并可增强其向成骨细胞分化,Cs A对AA患者BM-MSC向成骨细胞分化无明显促进作用。

This study was aimed to explore the regulation of arsenic trioxide（ As2O3） on imbalance between adipogenic and osteogenic differentiation of BM-MSC from patients with aplastic anemia（AA）.The BM-MSC from AA patients were separated and purifiedplaced into the adipogenic and osteogenic differentiation culture systemthen added the As, CsAAscombined with CsA were added to corresponding differentiation culture systemthe concentration of Asand CsA were set at 0.001 μ/ml and 2.5 mmol /ml respectivelythe cells were divided into Asgroupthe CsA groupcombined group and control group（ no drug）.The cell morphological observationoil red ＇O＇ stainingVon-Kossa stainingand RT-PCR were used to detect corresponding differentiation marks.The results indicated that in respect to adipogenic differentiationcellular morphology observing and oil red ＇O＇ staining showed that the rate of adipocyte differentiation in Asgroup was（ 18.3 ± 1.9）%which was lower than the（ 91.8 ± 2.7）% in the CsA group and（ 92.1 ± 1.2）% in control group（P〈0.05）,there was no significant difference in comparison with（ 8.3 ± 1.9）% in the combined group（ P〈0.05,but the rate of differentiation in CsA group was higher than that in combined group（ P〈0.05）,and there was no significant difference in comparison wtih control group.RT-PCR showed that the LPL-mRNA expression level in Asgroup were significantly lower than that in the CsA group and the control group（ P〈0.05）,no difference was observed while compared with the combined groupP〉0.05）,but the LPLmRNA expression level in CsA group was significantly higher than that in the combined group（ P〈0.05）.In terms of osteogenetic differentiationthe calcium deposition in Asgroup and combined group was obviously observed while rarely in the CsA group and the control group when detected by the Von-Kossa staining.OST-mRNA expression level in Asgroup were higher than that in CsA group and the control group（ P〈0.05）,while compared with the combined groupthere was no significant difference（P〉0.05）,but the OST-mRNA expression level in the CsA group was lower than that in the combined group（ P〈0.05）.It is concluded that Ascan significantly enhance the ability of BM-MSC from AA patients to differentiate into osteoblastalso can inhibit the adipogenic differentiationin contrastthe CsA can not promote the osteoblast differentiation of BM-MSC from AA patients.