再生障碍性贫血患者骨髓间充质干细胞转化生长因子β1及Smad3表达下调的发病学意义  

Significance of downregulated expression of transforming growth factor-β1 and Smad3 in bone marrow mesenchymal stem cells of the patients with aplastic anemia

作　　者：宋永平[1] 房佰俊[1] 张龚莉 林全德[1] 汪萍[1] 买玲[1] 魏旭东[1] 

机构地区：[1]河南省肿瘤医院血液科河南省血液病研究所,郑州450008

出　　处：《中国医药生物技术》2007年第4期276-280,共5页Chinese Medicinal Biotechnology

基　　金：河南省杰出青年基金(0612000900);河南省医学科技创新人才工程项目(200590)

摘　　要：目的研究骨髓间充质干细胞(MSC)中转化生长因子β1(TGF-β1)与Smad3的表达下调与再生障碍性贫血(再障)发生的可能关系。方法取20例重型再障患者骨髓MSC,分离制备出Flk1+CD34–MSC后进行以下试验:①体外定向诱导MSC向脂肪细胞分化,倒置显微镜观察成脂分化情况,油红O染色法鉴定脂肪细胞,用实时荧光定量PCR检测分化过程中脂蛋白酯酶(LPL)基因的表达;②用蛋白质印迹法检测MSC中TGF-β1及Smad3的表达,用ELISA检测MSC分泌TGF-β1的能力;③观察不同剂量(0、5、10、15、20ng/ml)TGF-β1对再障患者骨髓MSC成脂分化和增殖的影响。以7名健康成人的骨髓MSC相应检测为对照。结果再障患者骨髓MSC经4d培养后即分化为脂肪细胞,而健康成人骨髓MSC中仅见少量细胞出现脂肪滴。再障患者MSC培养4和8d时LPL基因表达水平分别为0.091±0.028、0.142±0.033,均高于健康成人骨髓MSC(分别为0.021±0.011及0.049±0.010,均P<0.01);而TGF-β1及Smad3表达水平明显低于健康成人骨髓MSC,TGF-β1分泌水平(3.4μg/L±0.9μg/L)也明显低于健康成人骨髓MSC(11.6μg/L±1.2μg/L,P<0.01)。在向脂肪细胞分化过程中,仅加入5ng/ml的TGF-β1即可明显抑制再障患者骨髓MSC向脂肪细胞定向诱导分化,同时促进其增殖。结论再障患者骨髓MSC中TGF-β1及Smad3表达水平的显著下调可能参与了再障的部分发病环节。Objective To study the relationship between the downregulated expression of transforming growth factor-β1 (TGF-β1) and Smad3 in bone marrow mesenchymal stem cells (MSC) and aplastic anemia (AA). Methods Flk1+CD34- MSC were separated from 20 patients with AA, then the following experiments were carried out: ①The Flk1+CD34- MSC were induced to differentiate into adipocytes in vitro. The differentiation were observed under an inverted microscope. Then the differentiated cells were identified by oil red O staining, and the expression of lipoprotein lipase (LPL) was evaluated using real-time fluorescent quantitative PCR. ②The levels of TGF-β1 and Smad3 expression in the MSC from patients with AA were investigated using western blot. The ability of the MSC to secrete TGF-β1 was determined by enzyme-linked immunosorbent assay (ELISA). ③ The effect of TGF-β1 in different doses on the proliferation and adipocytic differentiation of the MSC of the patients with AA was observed by using MTT. The bone marrow MSC obtained from 7 healthy adults were used as the control. Results After been cultured for 4 to 5 days, the bone marrow MSC from the patients with AA were filled with fat drops, while in the control group fat drops were observed in only a few MSC. After been cultured for 4 and 8 days, the levels of LPL expression in the MSC of patients with AA were 0.091 ± 0.028 and 0.142 ± 0.033 respectively, which were significantly higher than those in the MSC from the healthy adults (0.021 ± 0.011 and 0.049 ± 0.010 respectively, P < 0.01). The levels of TGF-β1 and Smad3 expression and TGF-β1 secretion in the MSC from the patients were significantly lower than those in the control (TGF-β1 secretion: 3.4 μg/L ± 0.9 μg/L vs 11.6 μg/L ± 1.2 μg/L). TGF-β1 at a dose of 5 ng/ml could markedly inhibit the differentiation of the MSC of AA patients into adipocytes and promote the proliferation of the cells. Conclusion The markedly downregulated expression of TGF-β1 and Smad3 in the MSC from AA patients may play an r

关 键 词：贫血 再生障碍性 骨髓 间质干细胞 转化生长因子分化β Smad3蛋白质 成脂分化 

分 类 号：R556[医药卫生—血液循环系统疾病]