骨髓源性肝样细胞门静脉移植大鼠肝纤维化模型

背景：研究已证明，骨髓间充质干细胞在体内、体外均可转化为肝细胞样细胞，并具有肝细胞的合成和分泌功能。 目的：将骨髓间充质干细胞诱导分化的肝样细胞移植到同种大鼠已纤维化的肝脏，观察其分布、分化情况及对肝功能的影响。 设计、时间及地点：随机对照动物实验，于2007-06/2008-03在泸州医学院附属医院实验室完成。 材料：100 g清洁级雄性SD大鼠10只用于分离培养骨髓间充质干细胞并诱导分化为肝样细胞。200~250 g清洁级雄性SD大鼠 75只，随机分为肝样细胞移植组30只，模型对照组30只，正常对照组15只。肝样细胞移植组及模型对照组大鼠腹腔注射四氯化碳制备肝纤维化动物模型。 方法：模型对照组和正常对照组大鼠门静脉输注不含血清的低糖DMEM培养基，肝样细胞移植组输入DAPI标记的肝样细胞，移植后第1，2，3，4周处死大鼠，留取肝脏和血清标本。 主要观察指标：肝组织Masson三重染色分期评估纤维化程度。肝脏冰冻切片荧光显微镜下观察肝样移植细胞定居情况。以全自动生化分析仪与放射免疫法检测血清肝功能指标和血清肝纤维化指标。 结果：肝样移植细胞逐渐从血管进入肝脏实质，最终散布于肝细胞间，移植3周后，荧光衰减明显。移植后第2周，肝组织中的假小叶逐渐吸收或消散，纤维化程度明显轻于模型对照组，胶原染色变浅，分布于纤维间隔和汇管区，肝细胞呈气球样变性。移植后第4周肝细胞排列接近正常，偶见点状坏死，无纤维间隔，血清肝功能指标和肝纤维化指标与正常对照组相比，差异无显著性意义(P > 0.05)。 结论：诱导的肝样细胞植入纤维化的肝组织后，细胞可停留于汇管区和肝细胞索，植入细胞最早可见于移植后第7天，血生化检查结果提示植入诱导的肝样细胞能够部分替代肝细胞的功能。

Transplantation of bone marrow-derived hepatocyte-like cells via the portal vein in rat hepatic fibrosis models

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can differentiate into hepatocyte-like cells in vivo and vitro and show hepatocyte function such as synthesis and secretion. OBJECTIVE: To induce and differentiate BMSCs into hepatic cells, which are transplanted into homogeneitic rat livers that had fibrous degenerated, and to observe the distribution, differentiation and effects of the hepatocytes on hepatic function. DESIGN, TIME AND SETTING: Randomized controlled animal experiment was performed at the Laboratory of Hospital Affiliated to Luzhou Medical College from June 2007 to March 2008. MATERIALS: A total of 10 clean male Sprague Dawley rats weighing 100 g were selected to culture BMSCs and to be induced and differentiated into hepatocyte-like cells. A total of 75 clean male Sprague Dawley rats weighing 200-250 g were randomly divided into hepatocyte-like cell transplantation group (n=30), model control group (n =30) and normal control group (n =15). Rats in the hepatocyte-like cell transplantation group and model control group intraperitonealy underwent carbon tetrachloride to prepare animal models of hepatic fibrosis. METHODS: Rats in the model control group and normal control group were subjected to serum-free low-glucose DMEM via portal vein infusion. Rats in the hepatocyte-like cell transplantation group received DAPI labelled hepatocyte-like cells. All rats were sacrificed to obtain liver and serum samples at 1, 2, 3 and 4 weeks following transplantation. MAIN OUTCOME MEASURES: Degree of the hepatic fibrosis was evaluated though Masson triple stain. Frozen sections were made to detect transformation of livers which had been transplanted cells with a fluorescence microscope. Serum the parameters of liver function and hepatic fibrosis were detected by the automatic biochemistry analyzer and radio-immunifaction method. RESULTS: Transplanted cells from the blood vessels gradually accessed into the liver, and eventually distributed between the liver cells scattered. But after three weeks of transplantation, fluorescence obviously decayed. At week 2, liver false lobular gradually absorbed or dissipated, and fibrosis significantly milder than the model control group, with collagen staining lighting and distributing in the fiber interval and portal area, and liver cells presented Balloon-like degeneration. At week 4, liver cells arranged in normal. Dot necrosis was occasionally found without fiber interval. No significant differences in the parameters of liver function and hepatic fibrosis were found compared with the normal control group(P > 0.05). CONCLUSION: Induced hepatocyte-like cells could engraft into fibrotic liver after transfusion as early as on the 7th day and localized in collection tube region and liver sinus. Results of biochemistry detection showed that the engrafted hepatocyte-like cells could substitute liver function partly.