骨髓间充质千细胞分离培养及在移植肝中定居能力的研究

目的体外分离培养并鉴定扩增大鼠骨髓间充质干细胞（MSC），观察MSC在肝移植受体内的定居能力。方法直接贴壁法培养大鼠MSC，绘制细胞生长曲线；流式细胞仪检测细胞表面标志。定向诱导MSC向成骨和成脂肪分化，鉴定其多向分化潜能。DAPI荧光标记MSC，由门静脉注入肝移植受体，取肝组织冰冻切片，观察其在移植肝内的定居情况。结果直接贴壁法成功分离培养MSC，并在传代培养中得以纯化扩增。传代周期约5d，可在体外传代20代以上，具有强大的增殖能力。流式细胞仪检测符合MSC表型。MSC可成功分化为成骨细胞和脂肪细胞，具有多向分化的能力。DAPI标记MSC的阳性率达100％。荧光显微镜观察。可见MSC定位于移植肝内。结论直接贴壁法分离培养MSC简单易行，获得MSC纯度高，生物学特性稳定；MSC可以在移植肝内存活并定居。

Isolation, Culture and Identification of the Bone Marrow Mesenchymal Stem Cells (MSC) in vitro and the Distribution of MSC in the Rat Transplantation Liver

Objective To isolate, culture and identify the rat bone marrow mesenchymal stem cells（MSC） and study the distribution of MSC after injection into portal vein in rats of orthotropic liver transplantation. Methods MSC were separated with direct anchoring method, and then they were amplified and cultured. The characters of the cell, such as morphology, cell growth curve, phenotype were demonstrated. MSC were identified using flow cytometry. The abilities of differentiate along adipocytic and osteoblastic were investigated. MSC were labeled with DAPI and were injected into portal vein in rats after orthotropic liver transplantation. The distribution of MSC was observed by fluorescence after frozen section. Results By direct anchoring method, MSC were gained after purification and proliferation, and passaged every five days. The cell population consisted of spindle-shaped cells with significant renewal capacity, stabilities of the biology as they were cultured until 20 passages. Flow cytometry test results meet the MSC phenotype. MSC can be differentiated into adipocyte and osteoblast cell. The positive rate DAPI reached 100%. MSC labeled with blue fluorescence can be found in livers of rats. Conclusion Direct anchoring method is simple and feasible when used to separate and culture MSC.Purified MSC with steady biological characters were gained. MSC can successfully reside in the liver of rats.