【摘要】 背景:骨髓基质干细胞移植对损伤脊髓有一定修复作用,较神经干细胞移植更为理想,但疗效并不稳定,可能与其移植微环境有关。目的:拟建立体外神经细胞微环境作用下骨髓基质干细胞的分化模型,观察其分化过程中蛋白表达的差异。设计、时间及地点:蛋白水平的观察对照实验,于 2005-07/2007-05 在哈尔滨医科大学基础医学院神经生物实验室完成。材料:Wistar 成年大鼠及新生胎鼠。方法:取新生 Wistar 胎鼠脊髓,以培养神经细胞。从成年 Wistar 大鼠骨髓中分离骨髓基质干细胞进行体外培养和增殖,应用红色荧光蛋白 PKH26 标记骨髓基质干细胞。骨髓基质干细胞、神经细胞单独培养组分别将骨髓基质干细胞、神经细胞单独培养,共培养组、分层联合培养组分别将标记的骨髓基质干细胞与神经细胞在体外共培养及在双层培养皿中联合培养。主要观察指标:培养 7 d 后收集细胞分别进行神经特异性烯醇化酶和胶质纤维酸性蛋白免疫荧光检测。应用 SELDI-TOF-MS 技术筛选骨髓基质干细胞向神经细胞分化过程中变化明显的相关蛋白进行分析。结果:骨髓基质干细胞与神经细胞共培养和双层联合培养 7 d 后,骨髓基质干细胞呈类似神经细胞形态。免疫荧光检测结果示,共培养组骨髓基质干细胞的神经特异性烯醇化酶和胶质纤维酸性蛋白阳性率明显高于分层联合培养组(P < 0.05),分层联合培养组明显高于单独培养对照组(P < 0.05)。骨髓基质干细胞在向神经细胞转化过程中有 5 种蛋白表达发生明显变化:在分层联合培养组TIP39_RAT 和 CALC_RAT 表达增加,为原表达量的 5.344 和 2.805 倍;INSL6_RAT,PNOC_RAT 和 PCSK1_RAT 表达下降,为原表达量的 0.380,0.499 和 0.437 倍。结论:在体外神经细胞微环境作用下,骨髓基质干细胞与神经细胞在共培养和双层联合培养时均能诱导分化成神经细胞,接触培养比非接触培养分化率高。骨髓基质干细胞在向神经细胞转化过程中与 5 种蛋白TIP39_RAT,CALC_RAT,INSL6_RAT,PNOC_RAT 和 PCSK1_RAT 密切相关。更多还原

【Abstract】 BACKGROUND: Bone marrow mesenchymal stem cell transplantation is superior to neural stem cell transplantation to repair spinal cord injury; however, the therapeutic effect is unstable and possibly related to microenvironment. OBJECTIVE: To study the differentiation of cultured in vitro bone marrow mesenchymal stem cells (BMSCs) into neurocytes by establishing a microenvironment and to observe differential expression of protein. DESIGN, TIME, AND SETTING: Observational contrast study was performed at the Laboratory of Neurobiology, Basic Medical College, Harbin Medical University from July 2005 to May 2007. MATERIALS: Adult Wistar rats and newborn fetal rats were used in this study. METHODS: Spinal cord was obtained from fetal rats to culture neurocytes. While, BMSCs were separated from bone marrow of adult rats, and they were then cultured in vitro, proliferated, and labeled with red fluorescin PKH26. BMSCs and neurocytes were individually cultured in the BMSCs group and the neurocyte group, respectively. In addition, BMSCs and neurocytes were co-cultured in vitro in double-layer culture dish in the co-culture group and the layered combination group, respectively. MAIN OUTCOME MEASURES: The obtained cells after 7-day culture were immunofluorescently detected by neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP). Surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) technique was used to analyze associated protein that was apparently changed during the differentiation from BMSCs into neurocytes. RESULTS: Seven days after co-culture, BMSCs were morphologically shared like neurocytes. Immunofluorescence indicated that NSE- and GFAP-positive ratios of BMSCs in the co-culture group were significantly higher than the layered combination group (P < 0.05); while, the ratios in the layered combination group were significantly higher than BMSCs alone group (P < 0.05). Five protein expressions were changed during the differentiation from BMSCs into neurocytes, for example, TIP39_RAT and CALC_RAT expressions increased in the layered combination group, which were 5.344 and 2.805 times as the primary expressions; INSL6_RAT, PNOC_RAT, and PCSK1_RAT expressions decreased, which were 0.380, 0.499, and 0.437 times as the primary expressions. CONCLUSION: By a microenvironment, both BMSCs and neurocytes in the co-culture and layered combination groups can differentiate into neuroblasts; while, contact differentiation ratio is higher than non-contract one. The differentiation is closely related to five proteins, including TIP39_RAT, CALC_RAT, INSL6_RAT, PNOC_RAT, and PCSK1_RAT.更多还原