摘要
目的培育表达绿色荧光蛋白(GFP)的裸小鼠,并探讨将其用于人胶质瘤干细胞(HGSCs)移植实验研究。
方法将C57BL/6J-GFP转基因小鼠与NC系裸小鼠进行交配,在继代时严格挑选都表达GFP的无毛雄鼠(♂)与有毛母鼠(♀)交配,通过肉眼、免疫组织化学和荧光显微镜等方法观察GFP在裸小鼠皮肤和脏器中的表达情况,继将HGSCs原位移植于表达GFP的裸小鼠,以观察移植瘤的生长情况。
结果传至8代的裸小鼠,包括脑在内的全身主要器官和细胞都表达GFP。对用于HGSCs原位移植的脑,能清晣辨认出不发光的瘤细胞和发光的宿主细胞。而瘤细胞经人特异白细胞抗体(HLA)连接红色荧光剂(PE)的免疫组织化学染色后,在共聚焦显微镜下可清晰显示与宿主脑细胞的组织学关系。
结论用免疫功能健全、表达GFP的转基因鼠与免疫功能缺陷的裸小鼠杂交,可获得脑等组织器官表达GFP的裸小鼠。对其进行HGSCs原位移植,因可清晰辨认移植瘤与宿主组织的关系而有望用于肿瘤组织重构的研究。
Objective To incubate nude mice which could stably express green fluoresent protein(GFP),then human glioma stem cells(HGSCs)were transplanted intracranially into NC nude mice with GFP expression.
Methods Transgenic C57BL/6J mouse expressing GFP was crossed with NC nude mouse, then the male nude mouse expressing GFP was crossed with the female pubescence mouse to obtain nude mouse with GFP expression, expression of GFP in the skin and organs of nude mice were evaluated under direct observation, by immunohistochemiscal and immunofluorescence assay. Accordingly HGSCs were transplanted orthotopically into the brains of nude mice expressing GFP to observe the biological characteristics of the intracranial growth of the transplanted tumor.
Results The organs and cells were all obviously expressed GFP in the nude mice propagated to 8th generation. Nonluminous tumor cells were distinguished from the host cell distinctly in the brains bearing tumor transplanted orthotopically. Microanatomy relationship was clearly showed between the brain cells of host and tumour cells with immunohistochemiscal assay, which stained the tumor cells with species-specific monoclonal antibody(HLA) coupled with red fluorescer (PE) through confocal microscopy .
Conclusions The nude mice expressing GFP were obtained by crossing the trangenic mice bearing naive immunity with NC nude mice. Orthotopic transplantation of human brain glioma may be used in the investigation on the tumour tissue reconstitution for the identification between the transplantation tumor and host tissue .
引文
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