Y染色体原位杂交在追踪间充质干细胞上的应用

Application of in situ Y chromosomal hybridization in the detection of mesenchymal stem cells

背景:间充质干细胞移植治疗多发性硬化症是一种被日渐关注的新型治疗手段。然而,间充质干细胞是通过穿越血脑屏障发挥细胞替代作用还是通过免疫抑制发挥治疗作用仍有待进一步验证。目的:建立稳定有效的Y染色体原位杂交方法,观察间充质干细胞在多发性硬化症模型小鼠中的分布。设计、时间及地点:观察性实验,于2007-09/2009-02在健康科学研究所分子免疫学实验室完成。材料:C57BL/6小鼠30只,6～8周龄,体质量15～20g,雌鼠用于建立实验性自身免疫性脑脊髓炎模型。方法:用地高辛标记了针对Y染色体的DNA探针,进行原位杂交验证探针的特异性和敏感性。再将从雄鼠中分离的间充质干细胞移植到雌性实验性自身免疫性脑脊髓炎小鼠体内,进行原位杂交。主要观察指标:光学和荧光显微镜观察间充质干细胞在实验性自身免疫性脑脊髓炎小鼠体内的分布情况。结果:实验确立了探针的Y染色体特异性和敏感性,进一步建立了有效的原位杂交方法。追踪到间充质干细胞在实验性自身免疫性脑脊髓炎小鼠的脊髓内分布极少,而外周免疫器官内有一定数量的分布,表明间充质干细胞可能是通过外周免疫调节发挥治疗作用。结论:在外周脾和淋巴结内有一定数量的间充质干细胞分布,而在中枢神经系统的脊髓内只有极少数的间充质干细胞。

BACKGROUND： Transplantation of mesenchymal stem cells （MSCs） to multiple sclerosis （MS） is a new developed treatment to be given more and more attention. However, whether the MSCs function by cell replacement after going cross the blood-brain barrior or immune suppression needs further confirmation. OBJECTIVE： To establish a steady and effective method of Y chromosome in situ hybridization （ISH） and to detect the distribution of MSCs in a mouse model with MS. DESIGN, TIME AND SETTING： The observational study was performed at the Laboratory of Molecular Immunology, Institute of Healthy Science from September 2007 to February 2009. MATERIALS： A total of 30 C57BL/6 mice, aged 6-8 weeks and weighing 15-20 g, were selected. Female mice were used to establish experimental autoimmune encephalomyelitis （EAE）. METHODS： Y chromosme specific DNA probe labled with digoxine （DIG） was designed and ISH was performed to confirm that the designed probe was Y chromosome specific and biologically sensitive. We transplanted the MSCs from male mice into the female EAE mice, and tracked the MSCs by Y chromosome ISH. MAIN OUTCOME MEASRUES： Distribution of MSCs in mice with EAE was observed under the optical and fluorescence microscope. RESULTS： The probe was confirmed to be Y chromosome specific and biologically sensitive. What＇s more, a steady and effective method of ISH was established. Some MSCs were detected in the periphery organs-spleen and lymph node of mice with EAE; seldom in spinal cord which indicated that MSCs might play its roles by immune suppression. CONCLUSION： Some MSCs were detected in the periphery organs--spleen and lymph node of mice with EAE, but seldom in spinal cord in the central nervous system.