脐血间充质干细胞在大鼠损伤肝脏迁徙途径的实验

Experimental study on the migration of umbilical cord blood mesenchymal stem cells in injured liver of rat model

目的：建立慢病毒载体转染增强型绿色荧光蛋白基因至人脐血间充质干细胞的实验体系,观察绿色荧光蛋白标记的人脐血间充质干细胞在急性肝坏死大鼠模型肝脏局部的迁徙途径。方法：采集新鲜人脐血,梯度离心及低血清培养基体外分离、培养人脐血间充质干细胞,以慢病毒为载体转染绿色荧光蛋白基因至人脐血间充质干细胞。CC l4橄榄油溶液腹腔注射建立急性肝坏死大鼠模型,将2.0~5.0×106绿色荧光蛋白标记的人脐血间充质干细胞肝脏局部移植给模型鼠,24、48、72 h、1周、2周和4周分别处死模型鼠,冰冻切片,荧光显微镜下观察移植细胞在肝脏局部的迁徙途径。结果：转染细胞荧光显微镜下呈现均一绿色荧光影像。细胞移植24 h内可见荧光信号由进针孔迁徙至汇管区,移植治疗48 h时移植细胞定居于汇管区,48 h后荧光信号向坏死病灶区域迁徙,1周后在肝脏坏死局部区域可见稳定的荧光信号。结论：本实验构建的绿色荧光蛋白转染人脐血间充质干细胞示踪体系稳定表达绿色荧光蛋白。经动物实验证明人脐血间充质干细胞肝脏局部移植给肝坏死大鼠模型后在肝脏局部经历了＂进针孔至汇管区＂,＂汇管区至病灶区＂的二次迁徙模式。

Aim：To establish experimental transfection system of enhanced green fluorescent protein gene to human umbilical cord blood mesenchymal stem cells（HUCBMSCs） and to observe migration of HUCBMSCs labeled with enhanced green fluorescent protein（EGFP） in the injuried liver of rat model. Methods： Cord blood mononuclear cells ware isolated by density gradient centrifugation,HUCBMSCs were cultured with low-serum medium in vitro.EGFP was transfected into HUCBMSCs through lentiviral vector.2.0～5.0×106 EGFP-labeled HUCBMSCs were injected into the liver of rat model with acute hepatic necrosis induced by CCl4.Migration of GFP-HUCBMSCs was traced through detecting fluorescent signal in liver frozen section 24 h,48 h,72 h,1 week,2 week,4 week post-transplantation.Results： The transfected cells exhibited uniform green fluorescence under fluorescence microscope images.24 hours after the transplantation of GFP-HUCBMSCs to rat model with acute hepatic necrosis,the fluorescent signal can be seen in the portal area of liver;48 hours after the transplantation,stronger fluorescence signal with disperse pattern can be observed along the portal area;one week after the transplantation,fluorescent signal of transplanted cells dispersed in liver necrosis area;four weeks after the transplantation,fluorescent signal was still persistent.Conclusion：The tracer in this experiment system expressed green fluorescent protein stably.The animal experiments demonstrated that transplanted cell encounter twice migration course： the first was from pinhole to the liver periportal area,the second was from periportal area to the necrotic area.