应用成人骨髓间充质干细胞建立人-猴肝脏嵌合体

Establishment of human-rhesus chimeric liver using adult bone marrow mesenchymal stem cells

背景:建立人骨髓间充质干细胞与猴的嵌合体肝脏对研究干细胞体内增殖与分化具有重要意义。目的:应用成人骨髓间充质干细胞建立人-猴肝脏嵌合体动物模型。方法:分离成人骨髓间充质干细胞,纯化并培养至6代,使细胞量大于5×108。转染绿色荧光蛋白基因标记后在B超引导下移植到妊娠10周的胎猴肝脏中,幼猴出生后1个月和3个月,穿刺取肝脏组织活检,切片后荧光显微镜观察带绿色荧光的人源细胞数量及分布,免疫组织化学染色法检测人白蛋白的表达。结果与结论:荧光显微镜观察发现,幼猴出生后1个月及3个月均发现有人源骨髓间充质干细胞在肝脏内存活,并发生迁移,分布趋向于集中。免疫组织化学检测发现,幼猴出生后1个月及3个月肝脏内有表达人白蛋白的细胞存在,分布与带有绿色荧光的细胞较为一致。提示应用成人骨髓间充质干细胞在猴胚胎早期能够建立人-猴肝脏嵌合体,人源性骨髓间充质干细胞可在猴肝中分化成具有合成白蛋白功能的细胞。

BACKGROUND： Human-mammal chimeric liver chimera has been a vital significance for the proliferation and differentiation of bone marrow mesenchymal stem cells. OBJECTIVE： To establish an animal model of human-rhesus chimeric liver using adult bone marrow mesenchymal stem cells. METHODS： Adult bone marrow mesenchymal stem cells were isolated, purified and cultured for the sixth generation. The number of bone marrow mesenchymal stem cells was no less than 5x108. Bone marrow mesenchymal stem cells labeled with green fluorescent protein were transplanted into the liver of the embryo rhesus with pregnancy of 10 weeks under guided by type-B ultrasound. At the 1st and 3rd months of birth, the liver tissue of the infant rhesus was taken for biopsy. After routine pathological section, histological specimens were observed under fluorescence microscope to confirm if there were adult bone marrow mesenchymal stem cells positive for green fluorescent protein and their distribution, and detected by immunohistochemical staining to identify if human albumin expressed in the liver of infant rhesus. RESULTS AND CONCLUSION： Fluorescence microscope observation indicated that at the 1st and 3rd monthsafter birth, there were surviving bone marrow mesenchymal stem cells derived from human with green fluorescence in the liver of infant rhesus, and these cells migrated to form more concentrated distribution. The immunohistochemical results demonstrated that functional liver cells expressing human albumin were observed in the liver of infant rhesus at the 1Stand 3~ months after birth, and their distribution was in accordance with bone marrow mesenchymal stem cells with green fluorescence. Human-rhesus chimeric liver can be established using adult bone marrow mesenchymal stern cells, which can generate functional liver cells in the liver of infant rhesus.