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目的:探讨骨髓源性肝脏干细胞是否参与肝癌前病变的形成,评价槲芪散对肝癌前病变的干预作用。方法:取80g体重的雄性Wistar大鼠的股骨和胫骨,用PBS冲洗骨髓,按密度梯度离心法原代分离培养骨髓间充质干细胞(mesenchymal stemcell,MSCs),用绿色荧光蛋白(GFP)作为追踪标记物将MSCs进行标记。将大鼠分为6组:即常规饲养组、模型组、模型+MSCs移植组、肝大部切除+MSCs移植组、槲芪散治疗组8g/kg、槲寄生总碱治疗组8g/kg。除正常对照组外,各组动物在行肝大部切除术后,选择剩余肝脏不同部位表面注射经GFP标记好的MSCs,用荧光显微镜观察GFP标记MSCs并计算标记率。用免疫荧光化学的方法检测肝脏内GFP标记的细胞。用组织化学染色的方法及Western Blot的方法分别检测大鼠肝脏-GT的表达及大鼠肝脏AFP的表达。结果:模型组及及模型+MSCs移植组的肝脏中-GT灶的数量及面积显著增多,且AFP的表达明显升高,以模型+MSCs移植组的变化更为明显,与槲芪散及槲寄生碱组比较有显著性差异。结论:MSCs参与了肝癌前病变的发生发展过程,而槲芪散及槲寄生总碱能够改善肝脏内环境,影响归巢于肝脏的MSCs的分化方向,诱导MSCs分化为正常肝细胞。
Objective: To investigate whether bone marrow-derived liver stem cells participate in the formation of precancerous lesions of hepatocellular carcinoma (HCC) and evaluate the intervention effect of Qizhuang San on precancerous lesions of liver cancer. Methods: The femur and tibia of male Wistar rats weighing 80g were washed with PBS, the bone marrow was washed with PBS, mesenchymal stem cells (MSCs) were isolated by density gradient centrifugation and traced with green fluorescent protein (GFP) Markers MSCs are labeled. The rats were divided into 6 groups: conventional feeding group, model group, model + MSCs transplantation group, subtotal liver transplantation + MSCs transplantation group, 8g / kg treatment group and 8g / kg total mistletoe treatment group. Except for the normal control group, GFP-labeled MSCs were injected on the surface of different parts of the remaining liver after partial hepatectomy. The GFP-labeled MSCs were observed by fluorescence microscopy and the labeling rate was calculated. Immunofluorescence was used to detect GFP-labeled cells in the liver. The expression of liver-GT and the expression of AFP in rat liver were detected by histochemical staining and Western Blot. Results: The number and area of -GTs in the liver of model group and model + MSCs transplantation group were significantly increased, and the expression of AFP was significantly increased. The changes of model and MSCs transplantation group were more obvious, Parasiticus group was significantly different. CONCLUSIONS: MSCs are involved in the pathogenesis of HCC precancerous lesions. However, total astragalus and mistletoe alkaloids can improve the internal environment of the liver, affect the differentiation direction of MSCs homing to the liver, and induce MSCs to differentiate into normal liver cells.