绿色荧光蛋白转基因雄鼠肝细胞移植重建雌鼠的造血功能研究

Transplantation of liver cells from GFP transgenic male mice to reconstruct hematopoietic function in female mice

目的用荧光雄鼠的肝细胞回输半致死量射线照射的C57BL雌鼠,观察肝细胞是否有重建造血功能的作用。方法用荧光雄鼠的肝细胞回输给半致死量射线照射的C57BL雌鼠,在移植后18、39和53 d剪鼠尾,采血并用CD4-PE和CD8-PE标记,再用氯化铵溶血,上流式细胞仪检测。移植87 d后杀鼠取骨髓用荧光原位杂交检测Y染色体阳性细胞的百分比,并取外周血、脾细胞、肝细胞和骨髓细胞检测绿色荧光蛋白(GFP)阳性细胞的百分比。结果回输肝细胞的雌鼠外周血中检测到GFP阳性细胞,同时检测到一部分GFP阳性细胞转化成了CD4+或CD8+T细胞,外周血中GFP阳性细胞的比例与荧光原位杂交分析的骨髓中的Y染色体阳性比例呈正相关。同时在鼠的脾细胞、肝细胞和骨髓细胞中都检测到了GFP阳性细胞。而脾细胞中检测到的GFP阳性细胞百分比高于其他细胞,相比有统计学意义(P=0.003)。结论成年鼠肝细胞含有一定量的造血干细胞,在移植鼠体内存活并分布在多脏器,重建了雌鼠的造血功能。

【Objective】 Whether liver cells have hematopoietic reconstruction function was investigated by infusion of liver cells of fluorescent male mice into half-lethally irradiated C57BL female mice.【Methods】 Liver cells from fluorescent male mice were transfused back to half-lethally irradiated C57BL female mice.At 18,39,53 days after transplantation,mice were cut tails to collect blood samples,and labeled with CD4-PE and CD8-PE,and then bloods were hemolyzed with ammonium chloride.Then cells were detected by flow cytometry.87 days after transplantation,the mice were sacrificed to collect bone marrow to be detected Y chromosome positive percentage by fluorescence in situ hybridization.GFP positive cells rate were detected in peripheral blood,spleen cells,liver cells and bone marrow cells of the sacrificed mice.【Results】 GFP-positive cells were detected in the peripheral blood of female mice infused with liver cells.Some GFP-positive cells converted into CD4＋ or CD8＋ T cells.The percentage of GFP-positive cells was positively correlated with the proportion of Y chromosome cells in bone marrow.GFP positive cells can also be detected in peripheral blood,spleen cells,liver cells and bone marrow cells of the sacrificed mice.The percentage of GFP-positive cells detected in the spleen cells was higher than other cells with a statistical significance（P =0.003）.【Conclusion】 The liver cells from adult mice contain a certain amount of hematopoietic stem cells.These cells survived in female mice and distributed in various organs,and reconstructed hematopoietic function.