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背景:胰岛移植是治疗Ⅰ型糖尿病的最有效的方法。然而,供体组织来源的匮乏限制了其应用。近来干细胞研究的进展表明,干细胞疗法可能解决这一问题。目的:采用活化素A和视黄酸诱导骨髓间充质干细胞分化,探讨骨髓间充质干细胞分化为胰岛素分泌细胞的可能性。设计:随机对照实验。单位:解放军军事医学科学院生物工程研究所。材料:实验于2004-11/2005-06在解放军军事医学科学院生物工程研究所完成。Sprague-Dawley大鼠6只,雄性,体质量150～160g,由解放军军事医学科学院实验动物中心提供。方法:无菌抽取大鼠股骨骨髓,采用密度梯度离心法分离骨髓间充质干细胞。传代细胞随机分为4组,高糖组、活化素A和视黄酸组、β-巯基乙醇组和阴性对照组。分别采用含有高糖、活化素A和视黄酸、β-巯基乙醇等刺激因素的条件培养基进行诱导。应用免疫细胞化学和反转录聚合酶链反应等方法对分化细胞表型进行检测。主要观察指标:检测胰岛素和胰高血糖素,以及胰岛素1mRNA的表达。结果:骨髓间充质干细胞经过诱导后,①在高糖诱导组中,有散在的胰岛素和胰高血糖素阳性反应细胞出现。②活化素A和视黄酸组及β-巯基乙醇组有较多的胰岛素和胰高血糖素阳性反应细胞出现,而且这些细胞形成类似胰岛样的结构。③高糖组,活化素A和视黄酸组,β-巯基乙醇组均可见胰岛素1mRNA的表达。④阴性对照组未见有胰岛素和胰高血糖素阳性反应细胞出现以及胰岛素1mRNA的表达。结论:实验建立的一种基于活化素A和视黄酸及其他成熟因子的一种诱导方案,成功将骨髓间充质干细胞诱导分化为胰岛素阳性反应细胞,并且形成类似胰岛样结构,但其诱导效率有待进一步提高。
Background: Islet transplantation is the most effective treatment for type 1 diabetes. However, the lack of donor tissue has limited its use. Recent advances in stem cell research have shown that stem cell therapy may solve this problem. OBJECTIVE: To induce the differentiation of bone marrow mesenchymal stem cells by activin A and retinoic acid, and to explore the possibility of bone marrow mesenchymal stem cells differentiating into insulin-secreting cells. Design: Randomized controlled experiment. Unit: PLA Academy of Military Medical Sciences Institute of Bioengineering. MATERIALS: Experiments were performed at Institute of Biological Engineering, PLA Military Academy of Medical Sciences from November 2004 to June 2005. 6 Sprague-Dawley rats, male, body weight 150 ~ 160g, provided by the Experimental Animal Center of PLA Academy of Military Medical Sciences. METHODS: Rat femur bone marrow was aseptically extracted and bone marrow mesenchymal stem cells were isolated by density gradient centrifugation. The passage cells were randomly divided into 4 groups, high glucose group, activin A and retinoic acid group, β-mercaptoethanol group and negative control group. Respectively using high glucose, activin A and retinoic acid, β-mercaptoethanol and other stimuli of the conditioned medium for induction. Immunocytochemistry and reverse transcription polymerase chain reaction and other methods to detect the phenotype of differentiated cells. MAIN OUTCOME MEASURES: Insulin and glucagon, as well as insulin 1 mRNA expression were measured. Results: Bone marrow-derived mesenchymal stem cells were induced in the presence of scattered insulin and glucagon-positive cells in the high glucose-induced group. ② Activin A and retinoic acid group and β-mercaptoethanol group had more insulin and glucagon positive reaction cells, and these cells formed a similar islet-like structure. ③ high glucose group, activin A and retinoic acid group, β-mercaptoethanol group can be seen insulin 1mRNA expression. ④ negative control group, no insulin and glucagon positive cells and insulin 1mRNA expression. CONCLUSION: An induction protocol based on activin A, retinoic acid and other mature factors was successfully established to induce the differentiation of bone marrow mesenchymal stem cells into insulin-positive cells and to form islet-like structure but induced Efficiency needs to be further improved.