骨髓间充质干细胞向神经元样细胞诱导分化过程中端粒酶的变

背景：已知端粒酶活性变化与组织工程应用的安全性密切相关，而目前端粒酶在骨髓间充质干细胞诱导分化过程中的变化及机制研究尚少。 目的： 观察体外人骨髓间充质干细胞向神经元样细胞诱导分化过程中端粒酶的变化。 设计、时间及地点：细胞学开放性实验，于2006-12/2007-11在吉林大学第三临床医院实验中心及基础医学院病理生物学教育部重点实验室完成。 材料：无菌条件下采集非血液系统疾病的志愿者髂骨骨髓，体外分离培养人骨髓间充质干细胞。 方法：取第3代培养的人骨髓间充质干细胞，应用二甲基亚砜/丁羟茴香醚（DMSO/BHA）联合诱导法诱导其向神经元样细胞分化。 主要观察指标：绘制体外培养的人骨髓间充质干细胞的生长曲线。免疫荧光及细胞化学染色法分别检测诱导后细胞的巢蛋白和尼氏体的表达，TRAP-ELISA方法检测人骨髓间充质干细胞诱导前后端粒酶活性的变化。 结果： 体外培养的人骨髓间充质干细胞在第3~8代生长较快,第10代以后细胞的生长能力明显减弱。经DMSO/BHA诱导分化后的细胞能够表达具有神经元特征的巢蛋白及尼氏体结构。此外，TRAP-ELISA结果显示诱导2 h细胞端粒酶活性变化不明显，当诱导6 h以后细胞端粒酶活性明显降低，诱导至24 h时，细胞端粒酶活性已近阴性（P < 0.05）。 结论：人骨髓间充质干细胞在体外成功诱导分化为神经元样细胞的过程中端粒酶活性逐渐降低直至消失。

Telomerase activity changes during the differentiation of bone marrow mesenchymal stem cells into neuron-like cells

BACKGROUND: It was reported that the changes in telomerase activities had close relationship with the application safe of tissue engineering. There were few researches about the telomerase activity and mechanism during the induction of human bone marrow mesenchymal stem cells (hBMSCs). OBJECTIVE: To evaluate the telomerase activity during the differentiation of in vitro hBMSCs into neuron-like cells. DESIGN, TIME AND SETTING: The cytology opening experiments were performed at the Experimental Center of Third Clinical Hospital of Jilin University and the Key Pathobiology Laboratory of Ministry of Education, College of Preclinical Medicine, Jilin University between December 2006 and November 2007. MATERIALS: Bone marrow was sterilely collected from the iliac bone of volunteers without hematological system diseases. hBMSCs were harvested in vitro. METHODS: The third passage of hBMSCs was differentiated into neuron-like cells induced by butylated hydroxyanisole and dimethyl sulfoxide. MAIN OUTCOME MEASURES: Growth curve of in vitro cultured hBMSCs was drawn. Immunofluorescence and immunocytochemistry were used to identify nestin and Nissl body expression. TRAP-ELISA was used to detect telomerase activity before and after hBMSCs induction. RESULTS: The third to eighth passage of in vitro cultured hBMSCs rapidly grew, and the tenth or later passage of BMSCs significantly reduced in growth speed. After induction of butylated hydroxyanisole and dimethyl sulfoxide, high expression of nestin and nissl body was detected in the differentiated cells. In addition, TRAP-ELISA showed telomerase activity of hMSCs was insignificant after 2 hours of induction, and then decreased after 6 hours of induction, and the activity was negative after 24 hours of induction (P < 0.05). CONCLUSION: Telomerase activity decreases, even disappears during the differentiation of hMSC into neuron-like cells.