不同移植途径对大鼠骨髓干细胞迁移至肝脏及分化的影响

目的 探讨门静脉、尾静脉和肝动脉3种移植途径下大鼠骨髓干细胞迁移至肝脏及向肝细胞分化的情况.方法 取30只正常SD大鼠,采用2-乙酰氨基芴和四氯化碳溶液灌胃,制成急性肝损伤模型,取其骨髓,以淋巴细胞分离液分离出干细胞,再用荧光染料(PKH26)进行标记.将此30只急性肝损伤大鼠分为3组,每组10只,门静脉输注组大鼠麻醉后,取腹部正中切口,经门静脉注入自体骨髓干细胞悬液0.4 ml(4×106个,下同);尾静脉输注组大鼠经尾静脉注人自体骨髓干细胞悬液0.4 ml;肝动脉输注组大鼠麻醉后,取腹部正中切口,将胃十二指肠动脉远端结扎,用动脉夹阻断肝总动脉血流,迅速在胃十二指肠动脉结扎点前方进针,注入自体骨髓干细胞悬液0.4 ml.骨髓干细胞移植后2周,取各组肝脏组织,制成冰冻切片,再以用异硫氰基荧光素标记的抗体进行免疫组织化学染色,检测其PKH26标记阳性细胞以及自蛋白和CK18的表达情况.结果 各组大鼠肝脏切片中均可见散在的PKH26染色阳性的红色荧光,门静脉输注组、尾静脉输注组及肝动脉输注组PKH26染色阳性细胞数分别为(58.0±2.67)个、(57.8±3.04)个及(58.3±3.52)个,三组间的差异无统计学意K(P>0.05).各组肝组织切片均广泛表达白蛋白及CK18(绿色荧光),荧光显微镜下可见同时发绿色荧光和红色荧光的细胞(绿色荧光与红色荧光相叠加而成的黄色荧光).结论 经门静脉、尾静脉和肝动脉注入的骨髓干细胞均能定植于肝脏,并在损伤的肝脏中分化为肝细胞,3种途径的效果相当.

Study on migration and differentiation of rat bone marrow stem cells to hepatocytes under three different routes of transplantation

Objective To observe the migration and differentiation of rat bone rnarmw stem cells to liver under three different routes of transplantation.Methods Intragastric administration was taken to 30 SD rats with CCl4-AAF by which the acute hepatic injured models were made.Bone marrow mononuclear cells which were lsolated from bone marrow with lymphocyte separating medium were labeled by PKH26.The rats were divided into 3 groups.The rats in portal vein-transDlanted group anesthetized were autotransplanted with 0.4 ml bone marrow stem cells suspension via portal vein.The rats in tail vein-transplanted group were autotransplanted with 0.4 ml bone marrow stam cells suspenslon via tail vein.The central sections were made on the abdomen of the rats in hepatic artery-transplanted group to expose the hepatic artery.The distal end of the gastroduodenal arterv was ligated and the blood flow of the common hepatic artery was cut by clamp 0.4 ml bone marrow stem cells suspension was injected In front of ligation of the gastroduodenal artery.Two weeks after transPlantion,the liver tissues were taken from the rats of every group and cut into frozen sections.The immunohistochemistry staining by fluorescein isothiocyanate was done to the sections for detection of the PKH26-labeled cells and the expression of albumin and CK18.Results Red fluorescence of PKH26 in the sections of every group was observed.The number of PKH26-labeled cells in the liver of portal vein-transplanted group,tail vein-transplanted group and hepatic artery-transplanted group was(58.0±2.67),(57.8±3.04)and(58.3±3.52)respectively.There was no significant difference between the groups(P＞0.05).The sections in every group expressed albumin and CK18(green fluorescence).The cells expressing red and green fluorescence at the same time were observed under fluorescence microscope(the yellow fluorescence=red fluorescence+the green fluorescence).Conclusion Under injured liver status,bone marrow stem cells of three groups can migrate to the liver and differentiate into hepatocytes.Migration has corresponding effects in three routes of transplantation.