兔肝纤维化模型建立与自体骨髓干细胞移植的疗效

背景：国内外已基于不同研究需要建立了多种肝纤维化动物模型方法，多采用大鼠为实验对象，但大鼠血容量少，不利于生化指标的检测。 目的：探讨容易重复而且可靠的肝纤维化模型制作方法，并通过骨髓干细胞自体肝门静脉移植给予治疗，判定其疗效和安全性。 设计、时间及地点：随机对照动物实验，于2004-09/2006-03在解放军昆明总医院干细胞与组织工程研究中心完成。 材料：肝功能检查正常的健康日本大耳白兔38只，体质量2.0~2.5㎏，随机分为2组：健康对照组8只，模 型 组30只。 方法：连续12周皮下注射硫代乙酰胺诱导兔肝纤维化，将造模成功的17只大鼠分为移植组与空白对照组。采集移植组兔骨髓，分离获得骨髓单个核细胞，加入肝细胞生长因子和粒单细胞集落刺激因子对骨髓干细胞进行诱导分化72 h，经肝门静脉自体移植。 主要观察指标：移植后4周通过病理与生化指标分析诱导后骨髓干细胞对肝纤维化的治疗作用。 结果：注射硫代乙酰胺12周时，病理切片显示肝小叶结构破坏，肝索排列紊乱，肝细胞脂肪变性，有少许坏死，间质纤维组织增生，有部分伸入小叶，有炎性细胞浸润，为肝纤维化症状。经兔肝门静脉移植骨髓干细胞4周后，总蛋白、清蛋白和白球蛋白比值均有所提高，总胆红素、直接胆红素、谷丙转氨酶和谷草转氨酶均有所降低，空白对照组也有改善，移植组肝纤维化病变较空白对照组改善程度明显。 结论：皮下注射硫代乙酰胺能成功诱导兔肝纤维化，且致死率低，容易重复；骨髓干细胞移植有治疗肝纤维化的作用，有助于肝组织结构恢复和改善肝功能。

Establishment of rabbit hepatic fibrosis models and the outcome of autologous bone marrow stem cell transplantation

BACKGROUND: Many methods of creating animal models of hepatic fibrosis have been established and these methods use rats as experimental materials. Rats have low blood volume, which is not useful for detection of biochemical indicator. OBJECTIVE: To investigate easily repetitive and reliable methods of establishing hepatic fibrosis models, and to treat by bone marrow stem cell transplantation via autologous portal vein, and to judge its outcome and safety. DESIGN, TIME AND SETTING: The randomized control animal experiment was performed at the Center of Stem Cells and Tissue Engineering Research, Kunming General Hospital of Chinese PLA from September 2004 to March 2006. MATERIALS: A total of 38 healthy big-eared Japanese rabbits weighing 2.0-2.5 kg, which had normal liver function test, were randomly assigned into a healthy control group (n=8) and a model group (n=30). METHODS: Rabbit models of hepatic fibrosis were created by subcutaneously infusing thioacetamide for successively 12 weeks. Seventeen success rat models were divided into a transplantation group and a blank control group. Rabbit bone marrow was collected to harvest bone marrow mononuclear cells. Bone marrow stem cells were incubated in hepatocyte growth factor and granulocyte colony stimulating factor for 72 hours. Autologous transplantation was performed via transhepatic portal vein. MAIN OUTCOME MEASURES: Therapeutic effect of bone marrow stem cells on hepatic fibrosis was analyzed by pathological and biochemical indicators 4 weeks after transplantation. RESULTS: At 12 weeks after infusing thioacetamide, pathological section showed that hepatic lobules were destroyed; hepatic cord was disordered; liver cells had adipose degeneration, with few necrotic cells; interstitial fibrous tissues were hyperplastic, and folioles in some fibrous tissues, inflammatory cell infiltration were detected. These were the symptom of hepatic fibrosis. At 4 weeks after bone marrow stem cell transplantation via rabbit hepatic portal vein, the levels of total protein, albumin and globulin were enhanced. Contents of total bilirubin, direct bilirubin, alanine aminotransferase and aspartate aminotransferase were decreased. There were some improvements in blank control group. Improvement in hepatic fibrosis was more significant in the transplantation group compared with the blank control group. CONCLUSION: Subcutaneously infused thioacetamide can successfully induce rabbit hepatic fibrosis, with a low fatality rate and easily to repeat. Bone marrow stem cell transplantation has an effect on treating hepatic fibrosis and is useful for the recovery and improvement of hepatic structure and function.