中药祛脂素对再生障碍性贫血患者骨髓间充质干细胞脂肪分化的干预

Interventional effect of Quzhisu on adipogenic differentiation of mesenchymal stem cells derived from aplastic anemia patients

背景：祛脂素为复方中药制剂，其化学成分包含维生素A、胡萝卜素、芸香甙及槐甙A、B、C和生物碱等，可抑制脂肪细胞分化成熟。骨髓间充质干细胞可定向分化为脂肪细胞，因此可将其作为阻止骨髓脂肪化、成为治疗再生障碍性贫血的一个可能靶点。目的：观察复方中药祛脂素对再生障碍性贫血骨髓间充质干细胞向脂肪细胞分化的干预效果。设计、时间及地点；细胞观察，于2006—06／2007—03在大连医科大学附属二院血液风湿科实验室完成。材料：骨髓间充质干细胞来自12例再生障碍性贫血患者。祛脂素为本科室辅助治疗再生障碍性贫血的科内中药验方，主要含五灵脂、槐花米和山楂，每毫升相当于原中药材1g的糊状药液。方法：抽取患者骨髓液，Ficoll＋贴壁法分离培养骨髓单个核细胞，达70％～80％融合后扩增传代。取传至第3代的骨髓间充质干细胞，分为2组：脂肪细胞诱导组向含有10％FBS的DMEM培养体系中加入终浓度为0．5μmol／L氢化可的松、1μmol／L地塞米松、10mg／L牛胰岛素；祛脂素组在其基础上加入终浓度为1g／L的祛脂素，培养14d。主要观察指标：计算脂肪细胞阳性率。采用RT-PCR及Western blowing法测定细胞内PPARγmRNA、蛋白水平的表达。结果：与脂肪细胞诱导组比较，祛脂素组脂肪细胞阳性率、细胞内PPARγmRNA水平、PPARγ蛋白水平均明显下降（t=18．6，P〈0．01：t=189．64，P〈0．01；t=15．93，P〈0．01）。结论：祛脂素对再生障碍性贫血的骨髓间充质干细胞脂肪分化具有抑制作用，通过减少脂肪细胞形成、降低脂肪细胞特异性标志PPARγ的转录及蛋白表达，帮助恢复骨髓造血功能。

BACKGROUND： Chinese herb compound Quzhisu contains many chemical compositions, such as vitamin A, carotene, rutoside and sophoricoside A, B, C, together with alkaloid. Quzhisu may inhibit the differentiation of adipocytes. Because of the differentiation into adipocytes, bone marrow mesenchymal stem cells can be a possible target to prevent bone marrow from adipogenesis and cure aplastic anemia. OBJECTIVE： To investigate the intervention effects of Chinese herb compound Quzhisu on adipogenic differentiation of mesenchymal stem cells derived from aplastic anemia patients. DESIGN, TIME AND SETTING： A cell observation was calried out in the Hematology and Rheumatology Laboratory of the Second Affiliated Hospital to Dalian Medical University （Dalian, Liaoning Province, China） from June 2006 to March 2007. MATERIALS： Bone marrow mesenchymal stem cells were derived from 12 patients with aplastic anemia. Quzhisu was a traditional Chinese medical prescription, and taken as the adjunctive therapy for aplastic anemia （China）. The main ingredients included Wulingzhi, Kuihuami and Shanzha. One milliliter pasty liquor was equal to 1 g original Chinese crude herbs. METHODS： Mesenchymal stem cells were isolated, and mononuclear cells were cultured with Ficoll plus attachment method. When the cells confluenced to 70%-80%, the passage culture commenced. Mesenchymai stem cells at the third passage was collected and divided into two groups： both adipocyte differentiation group and Quzhisu group were added with Ficortril at the final concentration of 0.5 μ mol/L, dexamethasone at the final concentration of 1 g mol/L and 10 mg/L bovine insulin into the DMEM containing 10% FBS. Moreover, Quzhisu at the final concentration of 1 g/L was given in the latter group. All cells were culture for 14 days. MAIN OUTCOME MEASURES： Positive rate of adipocytes developed from mesenchymal stem cells was calculated. The PPAR γ mRNA and protein expressions were detected with reverse transcription polymerase chain reaction and Western blotting. RESULTS： Compared-with adipocyte differentiation group, the positive rate of adipocytes, the expressions of PPAR Y mRNA and protein of Quzhisu group decreased significantly （t=18.6, P 〈 0.01; t=-189.64, P 〈 0.01; t=-15.93, P 〈 0.01）. CONCLUSION： Quzhisu can inhibit the adipogenic differentiation of mesenchymal stem cells derived from aplastic anemia patient＇s bone marrow. The related mechanism involves the reduction of adipocytes and the decreasing expression of PPAR γ mRNA and protein, which benefit to restore the haemopoiesis function of bone marrow.